CN101172099A

CN101172099A - Stable isotonic lyophilized protein formulation

Info

Publication number: CN101172099A
Application number: CNA2007101928065A
Authority: CN
Inventors: J·安德亚; J·L·克莱伦德; C·C·赫苏; X·M·拉姆; D·E·奥维卡谢尔; S·J·夏尔; J·Y·杨; S·S·吴
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 1995-07-27
Filing date: 1996-07-23
Publication date: 2008-05-07
Anticipated expiration: 2016-07-23
Also published as: AU715338B2; EP0841946A1; RU2229288C2; AU6638196A; JPH11510172A; JP4153560B2; AR003468A1; ZA966075B; CN101172100A; IL122733A; DK0841946T4; DE69624116T2; CA2226624C; ATE225185T1; ZA966240B; MX9800760A; WO1997004807A1; EP0841946B1; ES2186796T5; EP0841946B2

Abstract

The present invention describes a stable lyophilized protein formulation, which can be reconstituted with a suitable diluent to generate a high protein concentration reconstituted formulation which is suitable for subcutaneous administration. For example, anti-IgE and anti-HER2 antibody formulations have been prepared by lyophilizing these antibodies in the presence of a lyoprotectant. The lyophilized mixture thus formed is reconstituted to a high protein concentration without apparent loss of stability of the protein.

Description

The lyophilized protein formulation of stable isotonic

The application is that application number is 200410030256.3, and the applying date is on July 23rd, 1996, and denomination of invention is divided an application for the patent application of " lyophilized protein formulation of stable isotonic ".

Invention field

The present invention relates to a kind of freeze dried protein formulation.More specifically, the present invention relates to a kind of stable lyophilized protein formulation, its available diluent is rebuild and is generated a kind of preparation that is applicable to the stable reconstruction of subcutaneous administration.

Correlation technique

In in the past 10 years, development of biology makes can produce the various protein that are used for medicinal usage with recombinant DNA technology.Because protein is than organic and inorganic drug commonly used bigger more complicated (promptly also having a plurality of functional groups) except complicated three-D space structure is arranged, so these proteinic preparations have caused the problem that some are special.In order to make protein that biological activity be arranged, preparation must guarantee the conformation integrity of gal4 amino acid core sequence at least, and also wants the polyfunctional group of protected protein matter to prevent its degraded simultaneously.The approach of protein degradation comprises chemical instability (that is, any formation or fracture by key comes modifying protein to generate new chemical substance) or physical instability (that is the change of protein higher structure).Chemical instability can be caused by desamidation, racemization, hydrolysis, oxidation, β cancellation or disulfide exchange.Physical instability is to be caused by for example degeneration, cohesion, precipitation or absorption.Three kinds of modal proteolytic pathway are protein condenses, deacylated tRNA amine and oxidation (people such as Cleland, Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993)).

Lyophilization is the proteinic technology of a kind of preservation commonly used, and it can be removed the water in the protein articles of being concerned about.Lyophilization or freeze dried method are earlier that material to be dried is freezing, and distillation removes deicing or refrigerated solvent under vacuum environment then.Can comprise excipient in the preparation before lyophilizing, stability (the Pikal stable and/or raising freeze-drying prods when preservation with raising in freezing dry process, people such as M.Biopharm.3 (9) 26-30 (1990) and Arakawa, Pharm.Res.8 (3): 285-291 (1991)).

An object of the present invention is to provide a kind of lyophilized protein formulation stable when preservation and transportation.Also have a purpose to provide a kind of protein formulation that is used for the stable reconstruction of subcutaneous administration.In some example, an object of the present invention is to provide a kind of preparation of muptiple-use use, it is stable in to patient's time of administration at least.

Summary of the invention

Basis of the present invention is to find can make stable lyophilized protein formulation with a kind of freeze drying protectant (1yoprotectant) (preferably saccharide such as sucrose or trehalose); freeze dried preparation can be rebuild and generate stable reconstituted formulation; protein concentration in the preparation of the protein concentration of preparation before than lyophilizing high many (as; high approximately 2-40 doubly; be preferably high 3-10 doubly, better high 3-6 doubly).Specifically, if the preparation internal protein concentration before the lyophilizing is 5mg/mL or lower, then the protein concentration in the reconstituted formulation is generally 50mg/mL or more.High protein concentration like this is considered to be specially adapted to be used for when preparation the situation of subcutaneous administration in the reconstituted formulation.Although the protein concentration in the reconstituted formulation is very high, find that reconstituted formulation was stable (that is, protein does not show tangible or unacceptable chemistry or physical instability) at 2-8 ℃ in about at least 30 days.In some instances, reconstituted formulation is isoosmotic.Although used the freeze drying protectant of low concentration to ooze preparation, find that the protein in the lyophilized formulations is gone up stability and the integrity that has kept its physics and chemistry when lyophilizing and preservation substantially when rebuilding, to obtain this grade.

When (as the antibacterial that is used to inject, when diluent BWFI) was rebuild, reconstituted formulation can be used as the preparation of muptiple-use use with comprising antiseptic.This preparation is applicable to, for example comes treatment of chronic diseases when patient need often carry out subcutaneous protein administration.The advantage of muptiple-use use preparation is that it can more easily be used for the patient, can cut the waste by the whole uses of bottle intensive amount, and save very big cost for Producer, (has reduced charging and cost of transportation) because several doses of medicaments are installed in the bottle.

According to discovery described herein; one aspect of the present invention provides a kind of stable, isoosmotic reconstituted formulation; it comprises that content is at least about protein and the diluent of 50mg/mL; reconstituted formulation makes by the freeze-dried mixture of protein and freeze drying protectant, wherein the protein concentration in the reconstituted formulation than lyophilizing before in the mixture protein concentration high about 2-40 times.

In another example; the invention provides a kind of stable reconstituted formulation; it comprises that content is at least about antibody and the diluent of 50mg/mL; reconstituted formulation makes by the freeze-dried mixture of antibody and freeze drying protectant, wherein the antibody concentration in the reconstituted formulation than lyophilizing before in the mixture antibody concentration high about 2-40 times.

Protein concentration and isotonicity required in freeze drying protectant in the leading portion in the lyophilized formulations and the ratio of protein and for example selected protein and freeze drying protectant and the reconstituted formulation are relevant.When oozing reconstituted formulation with full length antibody (as protein) and trehalose or sucrose (as freeze drying protectant) preparation increased protein concentration etc., ratio for example can be that per 1 mole of antibody is about 100-1500 mole trehalose or sucrose.

Usually, the preparation (pre-lyophilized) before the lyophilizing of protein and freeze drying protectant can comprise that also a kind of buffer comes to provide suitable pH according to the protein of preparation for preparation.For this purpose, find the most handy following histidine buffering liquid, it has shown frozen-dried protective (lyoprotective) performance.

Preparation also can comprise surfactant (as polysorbate (polysorbate)), finds that it can reduce to rebuild proteinic cohesion and/or reduce particulate formation in the reconstituted formulation.Surfactant can add in preparation, lyophilized formulations and/or the reconstituted formulation (reconstituted formulation) before the lyophilizing (but preferably the preparation before the lyophilizing) as required.

The present invention also provides a kind of method that stable grade is oozed reconstituted formulation for preparing; it comprises the freeze-dried mixture of protein and freeze drying protectant is rebuild in diluent; make the protein concentration in the reconstituted formulation be at least 50mg/mL, wherein the protein concentration in the reconstituted formulation than lyophilizing before protein concentration in the mixture high about 2-40 times.

In also having an embodiment, the invention provides a kind of method for preparing a kind of preparation, comprising: (a) with the mixture lyophilizing of protein and freeze drying protectant; (b) mixture in the reconstruction procedures (a) in diluent, the preparation of make rebuilding are to wait to ooze stablely, and protein concentration is at least about 50mg/mL.For example, the protein concentration in the reconstituted formulation can be to be about 80mg/mL to about 300mg/mL.Usually, the protein concentration in the reconstituted formulation than lyophilizing before protein concentration in the mixture high about 2-40 times.

The present invention also provides a kind of product, and it comprises: the container that the freeze-dried mixture of protein and freeze drying protectant (a) is housed; (b) freeze-dried mixture is reconstructed into the explanation that protein concentration in the reconstituted formulation is at least about 50mg/mL in diluent.Product comprises that also another is equipped with the container of the diluent injection antibacterial (BWFI) of aromatic alcohol (as comprise).

The present invention also provides a kind of treatment mammiferous method, comprises the reconstituted formulation to administration treatment effective dose disclosed herein, and wherein mammal suffers from the disease that need treat with the protein in the preparation.For example, preparation can carry out subcutaneous administration.

Found the preparation before a kind of lyophilizing of useful Anti-HER 2 in the example that describes in detail below, it comprises that content is the anti-HER2 of about 5-40mg/mL (for example 20-30mg/mL) and content sucrose or the trehalose for about 10-100mN (for example 40-80mM), buffer (histidine for example, pH6 or succinate buffering night, pH5) and surfactant (for example polysorbate).Find that lyophilized formulations kept stable at 40 ℃ at least 3 months, kept stable at 30 ℃ at least 6 months.This anti-HER2 preparation can be reconstructed into the preparation that is applicable to intravenous administration with diluent, and it comprises the anti-HER2 that content is about 10-30mg/mL, and is keeping steady at least about 30 Tian Bao calmly at 2-8 ℃.When needing the Anti-HER 2 of higher concentration (for example, when subcutaneous defeated the passing when being mode to patient's administration of antibody), lyophilized formulations can be rebuild and generate stable protein concentration is 50mg/mL or more reconstituted formulation.

Here the preparation before a kind of required anti-IgE antibodies lyophilizing of Fa Xianing comprises content and is about the anti-IgE of 5-40mg/mL (as 20-30mg/mL) and sucrose or the trehalose that content is about 60-300mM (for example 80-300mM or 80-170mM), buffer (histidine preferably, pH6) and surfactant (for example polysorbate).Freeze dried anti-IgE can keep stable at 30 ℃ at least 1 year.Said preparation can be rebuild and generate the preparation that a kind of anti-IgE content is about 15-45mg/ml (for example 15-25mg/mL), and it is applicable to intravenous administration, and keeps stable at 2-8 ℃ at least 1 year.Perhaps, when needing the anti-IgE of higher concentration in the preparation, lyophilized formulations can be rebuild to generate the preparation of a kind of anti-IgE concentration 〉=50mg/mL.

The invention provides a kind of stable grade and ooze reconstituted formulation; it comprises protein and the diluent that content is at least about 50mg/mL; reconstituted formulation is that the freeze-dried mixture from protein and freeze drying protectant makes; wherein the protein concentration in the reconstituted formulation than lyophilizing before protein concentration in the mixture high about 2-40 times; preferred freeze drying protectant is sucrose or trehalose; also comprise buffer in the preferred described preparation; more preferably described buffer is that histidine or succinate cushion night, and preferred described preparation also comprises surfactant.

The present invention also provides above-mentioned preparation to be used to prepare the application for the treatment of mammiferous medicine, and wherein mammal suffers from the disease that need treat with protein in the preparation, and preferred described preparation is used for subcutaneous administration.

The present invention also provides a kind of stable reconstituted formulation; it comprises antibody and the diluent that content is at least about 50mg/mL; reconstituted formulation is that the freeze-dried mixture from antibody and freeze drying protectant makes; wherein in the reconstituted formulation antibody concentration than lyophilizing before in the mixture antibody concentration high about 2-40 times, antibody is anti-IgE antibodies or Anti-HER 2 in the preferred described preparation.Preferred described preparation is an isotonicity.

The present invention also provides a kind of method for preparing stable reconstituted formulation; be included in the mixture of rebuilding protein and freeze drying protectant in the diluent; make that protein concentration is at least about 50mg/mL in the reconstituted formulation, wherein the protein concentration in the reconstituted formulation than lyophilizing before protein concentration in the mixture high about 2-40 times.

The present invention also provides a kind of method for preparing a kind of preparation, comprises step:

(a) mixture to protein and freeze drying protectant carries out lyophilization; With

(b) freeze-dried mixture of reconstruction procedures (a) in diluent make that reconstituted formulation is an isotonicity, stable, and protein concentration is at least about 50mg/mL.Protein concentration in preferred this method in the reconstituted formulation is that about 80mg/mL is to about 300mg/mL, protein concentration in the preferred reconstruction preparation than lyophilizing before in the mixture the high about 2-40 of protein concentration doubly, and preferably in the whole freezing dry run lyophilization be maintained at about 15-30 ℃ in the shelf temperature and carry out.

The present invention also provides a kind of product, comprising:

(a) container of the freeze-dried mixture of protein and freeze drying protectant is housed; With

(b) in diluent, be reconstructed into the explanation that protein concentration in the reconstituted formulation is at least about 50mg/mL about freeze-dried mixture.

Preferred described product also comprises the container that another is equipped with diluent, and diluent is the injection antibacterial BWFI that comprises aromatic alcohol in the preferred described product.

The present invention also provides a kind of preparation that comprises the freeze-dried mixture of freeze drying protectant and monoclonal antibody, and wherein the mol ratio of freeze drying protectant and monoclonal antibody is that about 100 to 1500 moles of freeze drying protectants are than 1 mole of antibody.

The present invention also provides a kind of preparation, comprises the Anti-HER 2 that content is about 5-40mg/mL, and content is about sucrose or the trehalose of 10-100mM, and buffer and surfactant preferably also comprise filler.Preferred described preparation is freeze dried and stablized at least 6 months at 30 ℃ that also preferred described preparation is rebuild with diluent, makes that the antibody concentration in the reconstituted formulation is about 10-30mg/mL, and wherein reconstituted formulation was stablized about 30 days at least at 2-8 ℃.

The present invention also provides a kind of preparation, comprises the anti-IgE antibodies that content is about 5-40mg/mL, and content is about sucrose or the trehalose of 80-300mM, and buffer and surfactant, preferred described preparation are freeze dried and stablized at least 1 year at 30 ℃.

The present invention relates to:

1. a stable grade is oozed reconstituted formulation; it comprises protein and the diluent that content is at least 50mg/mL; reconstituted formulation is that the freeze-dried mixture from protein and freeze drying protectant makes; wherein freeze drying protectant and proteinic mol ratio are 100-600 mole freeze drying protectant in the mixture: 1 mole of protein, wherein the protein concentration in the reconstituted formulation than lyophilizing before in the mixture the high 2-40 of protein concentration doubly.

2. according to 1 described preparation, wherein freeze drying protectant is sucrose or trehalose.

3. according to 1 described preparation, wherein also comprise buffer.

4. according to 3 described preparations, wherein buffer is histidine or succinate.

5. according to each described preparation of 1-4, also comprise surfactant.

6. stable reconstituted formulation; it comprises antibody and the diluent that content is at least 50mg/mL; reconstituted formulation is that the freeze-dried mixture from antibody and freeze drying protectant makes; wherein the mol ratio of freeze drying protectant and antibody is a 100-600 mole freeze drying protectant in the mixture: 1 mole of antibody, wherein in the reconstituted formulation antibody concentration the high 2-40 of antibody concentration is doubly in the mixture before than lyophilizing.

7. according to 6 described preparations, wherein antibody is anti-IgE antibodies or Anti-HER 2.

8. according to 6 or 7 described preparations, it is an isotonicity.

9. method for preparing a kind of preparation comprises step:

(a) mixture to protein and freeze drying protectant carries out lyophilization, and wherein freeze drying protectant and proteinic mol ratio are 100-600 mole freeze drying protectant in this mixture: 1 mole of protein; With

(b) freeze-dried mixture of reconstruction procedures (a) in diluent makes that reconstituted formulation is an isotonicity, and is stable, and protein concentration is at least 50mg/mL.

10. according to 9 described methods, wherein the protein concentration in the reconstituted formulation than lyophilizing before in the mixture the high 2-40 of protein concentration doubly.

11. according to 10 described methods, wherein the protein concentration in the reconstituted formulation is 80mg/mL to 300mg/mL.

12. according to 10 or 11 described methods, wherein the protein concentration in the reconstituted formulation than lyophilizing before in the mixture the high 2-40 of protein concentration doubly.

13. according to 10 described methods, wherein lyophilization remains under 15-30 ℃ in the shelf temperature and carries out in the whole freezing dry run.

14. a product comprises:

(a) container that the freeze-dried mixture of protein and freeze drying protectant is housed, wherein freeze drying protectant and proteinic mol ratio are 100-600 mole freeze drying protectant in this mixture: 1 mole of protein; With

(b) in diluent, be reconstructed into the explanation that protein concentration in the reconstituted formulation is at least 50mg/mL about freeze-dried mixture.

15., also comprise the container that another is equipped with diluent according to 14 described products.

16. according to 15 described products, wherein diluent is the injection antibacterial that comprises aromatic alcohol.

17. a preparation that comprises the freeze-dried mixture of freeze drying protectant and antibody, wherein the mol ratio of freeze drying protectant and antibody is that 100 to 600 moles of freeze drying protectants are than 1 mole of antibody.

18.1 described preparation is used to prepare the application for the treatment of mammiferous medicine, wherein mammal suffers from the disease that need treat with protein in the preparation.

19. according to 18 described application, wherein preparation is used for subcutaneous administration.

20. a preparation comprises the Anti-HER 2 that content is 5-40mg/mL, content is sucrose or trehalose, buffer and the surfactant of 10-100mM.

21., also comprise a kind of filler according to 20 described preparations.

22. according to 20 or 21 described preparations, wherein preparation is freeze dried and stablized at least 6 months under 30 ℃.

23. according to 20 described preparations, preparation is rebuild with diluent, makes that the Anti-HER 2 concentration in the reconstituted formulation is 10-30mg/mL, wherein reconstituted formulation was stablized under 2-8 ℃ 30 days at least.

24. a preparation comprises the anti-IgE antibodies that content is 5-40mg/mL, content is sucrose or trehalose, buffer and the surfactant of 80-300mM.

25. according to 24 described preparations, it is freeze dried and stablized at least under 30 ℃ 1 year.

The accompanying drawing summary

Fig. 1 represents the influence of reconstruct volume to the stability of freeze dried rhuMAb HER2.Lyophilized formulations is that the preparation before the lyophilizing that comprises 25mg/mL protein, 60mM trehalose, 5mM sodium succinate pH5.0 and 0.01% polysorbas20 TM makes.Lyophilized cake is placed on 40 ℃ of incubations, uses the BWFI of 4.0mL (o) or 20.0mL (●) to rebuild then.With the component (fraction) of the whole protein in the non-degeneration size exclusion chromatographic determination reconstituted formulation, and determine according to peak area with respect to the non-denatured protein matter of the total peak area that comprises condensation product.

Fig. 2 has described the influence of trehalose concentration to the stability of lyophilizing rhuMAb HER2.25mg/mL protein is at the 5mM sodium succinate, pH5.0 (circle) or 5mM histidine, the lyophilizing to the trehalose of 200mM (1200 mol ratio) that is 60mM (360 mol ratio) of pH6.0 (square) and concentration.Freeze dried protein is placed on 40 ℃ of incubations 30 days (solid) or 91 days (hollow).After rebuilding freeze-dried protein, measure complete proteinic amount with 20mL BWFI.

Fig. 3 has described trehalose concentration for the influence of long-time stability that is deposited in 40 ℃ lyophilizing rhuMAb HER2.25mg/mL protein is at the 5mM sodium succinate, pH5.0,0.01% polysorbas20 ^TMWith (■) in the 60mM trehalose or 5mM histidine, pH6.0,0.01% polysorbas20 ^TMWith () lyophilizing in the 60mM trehalose or 21mg/mL protein at the 10mM sodium succinate, pH5.0, (●) lyophilizing in 0.2% polysorbas20 TM and the 250mM trehalose.Freeze dried protein is placed on 40 ℃ of incubations, rebuilds with 20mLBWFI then.Rebuild the back and measure complete proteinic amount.

Fig. 4 is illustrated in 38.4mM mannitol (7mg/mL), 20.4mM sucrose (7mg/mL), 5mM histidine, pH6.0,0.01% polysorbas20 ^TMIn freeze dried rhuMAb HER2 stability.Freeze-dried protein uses the BWFI of 4.0mL (o) or 20.0mL (●) to rebuild at 40 ℃ of incubations then.Rebuild the back and measure complete proteinic amount.

Fig. 5 represents that rhuMAb HER2 is at 5mM sodium succinate, pH5.0,60mM trehalose, 0.01% polysorbas20 ^TMThe stability of rebuilding after the middle lyophilizing.Sample BWFI (the 20mL:0.9% benzyl alcohol of 4.0mL (square) or 20.0ml (circle); The 4mL:1.1% benzyl alcohol) rebuilds, be deposited in then under 5 ℃ (solid) or 25 ℃ (hollow).Non-denatured protein matter % determines that by the proteinic peak area of non-degeneration (native) (not degraded) with respect to total peak area peak area is measured with cation-exchange chromatography.

Fig. 6 represents the stability that rhuMAb HER2 rebuilds after the lyophilizing in 5mM histidine, pH6.0,60mM trehalose, 0.01% polysorbas20.Sample BWFI (the 20mL:0.9% benzyl alcohol of 4.0mL (square) or 20.0ml (circle); The 4mL:1.1% benzyl alcohol) rebuilds, be deposited in then under 5 ℃ (solid) or 25 ℃ (hollow).Non-denatured protein matter % determines that by the proteinic peak area of non-degeneration (not degraded) with respect to total peak area peak area is measured with cation-exchange chromatography.

Fig. 7 represents the stability that rhuMAb HER2 rebuilds after the lyophilizing in 5mM histidine, pH6.0,38.4mM mannitol, 20.4mM sucrose, 0.01% polysorbas20.Sample BWFI (the 20mL:0.9% benzyl alcohol of 4.0mL (square) or 20.0ml (circle); The 4mL:1.1% benzyl alcohol) rebuilds, be deposited in then under 5 ℃ (solid) or 25 ℃ (hollow).Non-denatured protein matter % determines that by the proteinic peak area of non-degeneration (not degraded) with respect to total peak area peak area is measured with cation-exchange chromatography.

Fig. 8 represents the stability that rhuMAb HER2 rebuilds after the lyophilizing in 10mM sodium succinate, pH5.0,250mM trehalose, 0.2% polysorbas20.Sample is rebuild with 20.0mL BWFI (0.9% benzyl alcohol), is deposited in then under 5 ℃ (●) or 25 ℃ (zero).Non-denatured protein matter % determines that by the proteinic peak area of non-degeneration (not degraded) with respect to total peak area peak area is measured with cation-exchange chromatography.

Fig. 9 represents that it is that 10mM, antibody concentration are the cohesion situation in the buffer of 5mg/mL that rhuMAb E25 adds pH5 to pH7, buffer concentration.The lyophilizing sample is also measured the cohesion after 0,4 weeks, 8 weeks and 52 weeks when being deposited in 2-8 ℃.Buffer is: potassium phosphate pH7.0 (zero); Sodium phosphate pH7.0 (); Histidine pH7.0 (◇); Sodium succinate pH6.5 (●); Sodium succinate pH6.0 (■); Sodium succinate pH5.5 (◆) and sodium succinate pH5.0 (▲).

Figure 10 is illustrated in the cohesion situation that freeze dried rhuMAb E25 in the 5mM histidine buffering liquid measures during in following preservation when pH6 and pH7.Buffer be for: pH6.0 is deposited in 2-8 ℃ (zero); PH6 is deposited in 25 ℃ (); PH6 is deposited in 40 ℃ (◇); PH7 is deposited in 2-8 ℃ (●); PH7 is deposited in 25 ℃ (■); And pH7, be deposited in 40 ℃ (◆).

Figure 11 is illustrated in the 10mM sodium succinate of pH5 and the frozen-dried protective agent concentration of adding is the cohesion situation of the 5mg/mL rhuMAb E25 of preparation among the 275mM (isoosmotic).Freeze drying protectant is: contrast does not contain freeze drying protectant (zero); Mannitol (); Lactose (◇); Maltose (●); Trehalose (■); And sucrose (◆).The lyophilizing sample and be determined at 0,4 weeks of 2-8 ℃ of preservation, 8 weeks and 52 weeks after the cohesion situation.

Figure 12 is illustrated in the 10mM sodium succinate of pH5 and the frozen-dried protective agent concentration of adding is the cohesion situation of the 5mg/mL rhuMAb E25 of preparation among the 275mM (isoosmotic).Freeze drying protectant is: contrast does not contain freeze drying protectant (zero); Mannitol (); Lactose (◇); Maltose (●); Trehalose (■); And sucrose (◆).The lyophilizing sample and be determined at 0,4 weeks of 40 ℃ of preservations, 8 weeks and 52 weeks after the cohesion situation.

Figure 13 represents rhuMAb E25 lyophilizing in the histidine buffering liquid of lactose isotonic concentration (being 275mM), pH6 of 20mg/mL, rebuilds the hydrophobic interaction chromatography result to 20mg/mL after 2-8 ℃, 25 ℃ or 40 ℃ of 24 weeks of preservation.

Figure 14 represents rhuMAb E25 lyophilizing in the histidine buffering liquid of pH6 of 20mg/mL, rebuilds the hydrophobic interaction chromatography result to 20mg/mL after 2-8 ℃, 25 ℃ or 40 ℃ of 24 weeks of preservation.

Figure 15 represents rhuMAb E25 lyophilizing in the histidine buffering liquid of sucrose isotonic concentration (being 275mM), pH6 of 20mg/mL, rebuilds the hydrophobic interaction chromatography result to 20mg/mL after 2-8 ℃, 25 ℃ or 40 ℃ of 24 weeks of preservation.

Figure 16 represents the influence of sugared concentration for the 20mg/mL rhuMAbE25 of preparation in 5mM histidine (pH6.0).Sucrose (●) and trehalose () add in the preparation (seeing the following form 1) with the mol ratio of 0 to 2010 (wait and ooze).The lyophilizing sample is determined at 50 ℃ of cohesion situations after 12 weeks of preservation.

Table 1

The mol ratio of sugar and E25 antibody	Sugar concentration (mM)
The mol ratio of sugar and E25 antibody	Sugar concentration (mM)	0	0
260	34.4	0	0
260	34.4	380	51.6
510	68.8	380	51.6
510	68.8	760	103.1
1020	137.5	760	103.1
1020	137.5	1530	206.3
2010	275	1530	206.3

Figure 17 is disclosed in 5mM histidine (pH6), 85mM sucrose (zero); 85mM trehalose (); Be mixed with the cohesion situation of 25mg/mL rhuMAb E25 in 161mM sucrose (◆) or the 161mM trehalose (▲).The lyophilizing sample also is deposited in 2-8 ℃, then with 0.9% benzyl alcohol be reconstructed into antibody concentration be 100mg/mL, histidine be 20mM, pH6 etc. ooze (340mM) and height oozes (644mM) sugared concentration preparation.

Figure 18 is disclosed in 5mM histidine (pH6), 85mM sucrose (zero); 85mM trehalose (); Be mixed with the cohesion situation of 25mg/mL rhuMAb E25 in 161mM sucrose (◆) or the 161mM trehalose (▲).The lyophilizing sample also is deposited in 30 ℃, then with 0.9% benzyl alcohol be reconstructed into antibody concentration be 100mg/mL, histidine be 20mM, pH6 etc. ooze (340mM) and height oozes (644mM) sugared concentration preparation.

Figure 19 is disclosed in 5mM histidine (pH6), 85mM sucrose (zero); 85mM trehalose (); Be mixed with the cohesion situation of 25mg/mL rhuMAb E25 in 161mM sucrose (◆) or the 161mM trehalose (▲).The lyophilizing sample also is deposited in 50 ℃, then with 0.9% benzyl alcohol be reconstructed into antibody concentration be 100mg/mL, histidine be 20mM, pH6 etc. ooze (340mM) and height oozes the preparation of (644mM) sugared concentration.

DESCRIPTION OF THE PREFERRED

I. definition

" protein " is meant that aminoacid sequence, chain length are enough to produce high-caliber tertiary structure and/or quarternary structure.This is not have the difference of the small-molecular weight medicine of this structure with " polypeptide " or other.Usually, the proteinic molecular weight here is at least about 15-20kD, preferably is at least about 20kD.

Here defining included proteinic example has mammalian proteins matter, and for example, growth hormone comprises human growth hormone and bovine growth hormone; Somatotropin releasing factor; Parathyroid hormone; Thyrotropin; Lipoprotein; α-1-antitrypsin; The INSULIN A chain; Insulin B chain; Proinsulin; Follicle-stimulating hormone; Calcitonin; Lutropin; Glucagon; Thrombin is as Factor IX C, factors IX, the tectotype factor and vWF ELISA; Anticoagulin such as protein C; Atrial natriuretic peptide; Curosurf; The plasminogen activator is as urokinase or tissue plasminogen activator (t-PA); Bombesin; Thrombin; Tumor necrosis factor-alpha and-β; Enkephalinase; Chemotactic factor RANTES (the activation back is adjustable usually by T cellular expression and secretion); Human macrophage inflammatory protein (MIP-1-α); Serum albumin such as human serum albumin; Mullerian inhibitory substance; Relaxin A chain; Relaxin B chain; Relaxation precipitinogen; The Mus gonadotropin is followed peptide; Deoxyribonuclease; Inhibin; Activin; VEGF (VEGF); The receptor of hormone or somatomedin; Integrin; A-protein or D; Rheumatoid factor; Neurotrophic factor such as bone spread out neurotrophic factor (BDNF), neurotrophin-3 ,-4 ,-5 or-6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factor such as NGN-β; Platelet derived growth factor (PDGF); Fibroblast growth factor such as aFGF and bFGF; Epidermal growth factor (EGF); Transforming growth factor (TGF) comprises TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 or TGF-β 5 as TGF-α and TGF-β; Insulin like growth factor-1 and-II (IGF-I and IGF-II); Des (1-3)-IGF-I (brain IGF-I); Insulin-like growth factor binding proteins; CD protein is as CD3, CD4, CD8, CD19 and CD20; Erythropoietin (EPO); TP (TPO); The osteogenic induction factor (osteoinductive factors); Immunotoxin; Bone morphogenetic protein (BMP); Interferon, as interferon-' alpha ' ,-β and-γ; Colony stimulating factor (CSF), for example M-CSF, GM-CSF and G-CSF; Interleukin (IL) is as IL-1 to IL-10; Superoxide dismutase; TXi Baoshouti; Membrane surface protein; Decay accelerating factor (DAF); Viral antigen, for example the part bag quilt of AIDS; Transport protein; Homing receptor; Addressin; Regulate albumen; Immunoadhesin; Antibody; Bioactive fragment or variant with any aforementioned polypeptides.

The protein of preparation is preferably pure basically, preferably is homogeneous (promptly not having foreign protein etc.) basically.The protein of " pure basically " refers to component and comprises protein at least about 90% (weight) (in the gross weight of component), preferable 95% (weight) that is at least about.The protein of " homogeneous phase basically " refers to the gross weight in component, and component comprises the protein at least about 99% (weight).

In some instances, protein is antibody.For example antibody can combine with above-mentioned arbitrary molecule.The typical target molecule of antibody comprises CD protein among the present invention, as CD3, CD4, CD8, CD19, CD20 and CD34; The member of HER receptor family, for example EGF receptor, HER2, HER3 or HER4 receptor; Cell adhesion molecule as LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM and α v/ β 3 integrins, comprises its α or β subunit (as, anti--CD11a, anti--CD18 or anti-CD11b antibody); Growth factors such as VEGF; IgE; Blood group antigen; The flk2/flt3 receptor; Greasiness (OB) receptor; Protein C etc.

The implication of term " antibody " is very wide, it comprises monoclonal antibody (including the full length antibody in immunoglobulin fc region territory), the antibody compositions that a plurality of epitope specificities are arranged, bi-specific antibody, double antibody (diabodies) and single chain molecule especially, and antibody fragment is (as Fab, F (ab ') ₂And Fv).

Terminology used here " monoclonal antibody " refers to the antibody that obtains from the antibody population of homogeneous basically, each antibody that promptly is included in this colony is identical, except on a small quantity natural sudden change taking place.Monoclonal antibody has the specificity of height to single antigen site.And, with (polyclone) antibody preparations of routine, promptly generally include at the prepared product of the different antibodies of different determinants (epi-position) on the contrary, each monoclonal antibody is only at the single determinant of antigen.Except specificity, the advantage of monoclonal antibody is that they are next synthetic with the hybridoma incubation, can not polluted by other immunoglobulin.Qualifier " monoclonal " refers to the feature of the antibody that obtains from the antibody population of homogeneous basically, need should not be understood that any special method produce antibody.For example, the used monoclonal antibody of the present invention can prepare with the hybridoma method (Nature, 256:495 (1975)) that people such as Kohler describe, or prepares (seeing U.S. Patent No. 4,816,567) with recombinant DNA technology." monoclonal antibody " also available technology of describing as people (J.Mol.Biol.222:581-597 (1997)) such as people such as Clackson (Nature, 352:624-628 (1991)) and Marks is separated acquisition from phage antibody library.

The monoclonal antibody here is particularly including " chimeric " antibody (immunoglobulin), its part heavy chain and/or light chain are identical or homology with the corresponding sequence of antibody that obtains or belong to specific antibodies class or subclass of deriving from particular types, and remaining chain is identical or homologous with derive from another kind of apoplexy due to endogenous wind antibody acquisition or that belong to another antibody class or subclass and the segmental corresponding sequence of this antibody, as long as they show required biological activity (U.S. Patent No. 4,816,567; People such as Morrison, Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984)).

" humanization " antibody in non-human body source (as Mus source type) is that gomphosis immunoglobulin, immunoglobulin chain or the fragment that comprises the minmal sequence that obtains from the type immunoglobulin of inhuman source are (as Fv, Fab, Fab ', F (ab ') ₂Or other and the bonded antibody sequence of antigen).The major part of humanized antibody is a people source type immunoglobulin (receptor antibody), and wherein the residue of the complementary determining region of receptor (CDR) is substituted by the CDR residue of the antibody that required specificity, affinity and ability are arranged of inhuman source type such as Mus, rat or rabbit antibody (donor antibody).In some instances, Fv framework region (FR) residue of people source type immunoglobulin is replaced by corresponding inhuman source type residue.And humanized antibody can be included in the residue that does not all have in the CDR of receptor antibody and input or the frame sequence.Further refine and optimize the performance of antibody of these modifications.Usually, humanized antibody all comprises at least one basically, typically comprise two Variable Areas, wherein all or all basically CDR district corresponding with inhuman source type immunoglobulin and all or all basically FR districts are the sequences of people source type immunoglobulin.Humanized antibody can also randomly comprise partial immunity globulin at least, normally the constant region (Fc) of people source type immunoglobulin.More detailed description is seen the description that people such as Jones are done at Nature (332:323-329 (1988)) and Presta people such as Nature (321:522-525 (1986)), Reichmann in Curr.Op.Struct.Biol. (2:593-596 (1992)).Humanized antibody comprises Primatized ^TMAntibody, wherein the antigen binding domain of antibody is by obtaining in the antibody of Macaca (macaque) monkey immunity inoculation being gone into interested antigen and producing.

The preparation of " stablizing " is meant that protein wherein can keep its physics and chemical stability and integrity basically when preservation.The analytical technology of existing multiple mensuration protein stability in this field, they are at " peptide and pharmaceutical grade protein transmission " (Peptide and Protein Drug Delivery, 247-301, VincentLee edits, Marcel Dekker Inc., New York, New York publishing house (1991)) and Jones summary to some extent in A.Adv.Drug Delivery Rev. (10:29-90 (1993)).Stability can be measured in chosen temperature with in the time.For carrying out rapid screening, preparation can and be measured stability simultaneously in 40 ℃ of 2 weeks to 1 of preservation month.When preparation will be deposited in 2-8 ℃, preparation should be stablized 1 month and/or stablize at least 2 years at 2-8 ℃ at least at 30 ℃ or 40 ℃ usually.When preparation will be deposited in 30 ℃, preparation should be stablized 2 years and/or stablize at least 6 months at 40 ℃ at least at 30 ℃ usually.For example the cohesion degree after lyophilizing and preservation can be used as a sign (seeing the example here) of protein stability.For example, the preparation of " stablizing " be in the preparation less than about 10%, being preferably less than about 5% protein is the form of condensation product.In another example, after the lyophilizing of lyophilized formulations and preservation, measured the increment of preparation condensation product.For example, the lyophilized formulations of " stablizing " be meant when lyophilized formulations 2-8 ℃ of preservation in the time of at least 1 year, the condensation product increment should be less than about 5%, preferably less than about 3% in the lyophilized formulations.In another example, the stable available biological activity determination method of protein formulation is measured (face embodiment 2 as follows).

" reconstruction " preparation is meant by lyophilized protein formulation is dissolved in protein is dispersed in the reconstituted formulation.In some examples of the invention, be applicable to that reconstituted formulation to the patient's administration (as the parenteral administration) that need treat with required protein is applicable to subcutaneous administration.

" wait and ooze " and refer to that interested preparation equates with the osmotic pressure of human blood basically.Be generally about 250 to 350mOsm Deng the osmotic pressure that oozes preparation.Isotonicity can be with measuring as vapour-pressure type or freezing type permeability manometer.

" freeze drying protectant " refers to a kind of molecule, when it combines with protein of interest matter, can stop or reduce chemistry and/or the physical instability of protein when lyophilizing and follow-up preservation.Typical freeze drying protectant comprises saccharide such as sucrose or trehalose; Aminoacid such as monosodium glutamate or histidine; Methylamine class such as betanin (betaine); The salt of Yi Rong such as magnesium sulfate; Polyhydric alcohol such as trihydroxylic alcohol or senior sugar alcohol are as glycerol, erithritol, glycerol, arabitol, xylitol, Sorbitol and mannitol; Propylene glycol; Polyethylene Glycol; Pluronic; And combination.Preferable freeze drying protectant is a nonreducing sugar, as trehalose or sucrose.

When amount " frozen-dried protective amount " the finger protein matter in the preparation before the freeze drying protectant adding lyophilizing exists at the freeze drying protectant of frozen-dried protective amount, when lyophilizing and preservation, keep its physics and chemical stability and integrity basically.

Here interested " diluent " is pharmaceutically acceptable (administration is a safety non-toxic for human body), can be used to prepare reconstituted formulation.Typical diluent comprises sterilized water, injection antibacterial (BWFI), pH buffer (as phosphate buffer), aseptic salt solution, Ringer's solution (Ringer ' ssolution) or Glucose Liquid.

" antiseptic " is a kind of the adding in the diluent reducing the chemical compound of the bacterial action in the reconstituted formulation basically, thereby can obtain muptiple-use use reconstituted formulation.Effectively examples of preservatives comprises chlorination octadecyl 3,5-dimethylphenyl ammonium, chlorination hexane first ammonium, Benzene Chloride alkane _ (mixture of chlorination alkyl phenyl Dimethyl Ammonium, wherein alkyl is a long-chain compound) and chlorination Benzethonium.The antiseptic of other type comprises aromatic alcohol such as phenol, butanols and benzyl alcohol, alkyl paraben such as methyl or propyl group paraben, catechol, resorcinol, cyclohexane extraction, 3-amylalcohol and metacresol.Here Zui Jia antiseptic is a benzyl alcohol.

" filler " is the chemical compound that forms lyophilized cake physical arrangement (as it is easier that the lyophilized cake of keeping open-celled structure of homogeneous is basically produced) in a kind of adding freeze-dried mixture.Typical filler comprises mannitol, glycerol.Polyethylene Glycol and Sorbitol.

" treatment " refers to medical therapy and preventive measure.Those persons of benefiting from that need treat comprise suffering from disease and the prevent disease person of benefiting from.

Need " mammal " of treatment to refer to any mammiferous animal that classifies as, comprise people, domestic animal and farm-animals and zoo, sports ground animal or house pet, as Canis familiaris L., horse, cat, cattle etc.Preferable mammal is the people.

" disease " is to have benefited from the disease of protein therapeutic.It comprises chronic and acute illness or is in the disease that makes mammal be easy to fall ill of morbid state.Here nonrestrictive disease example to be treated comprises cancer and anaphylaxis.

II. working of an invention scheme

A. protein preparation

The technology that protein to be prepared has been set up with this field prepares, and they comprise the synthetic technology combination of these technology (synthetic as recombinant technique and peptide or) or separate from endogenous protein.In some examples of the invention, selected protein is antibody.The technology that produces antibody is as follows.

(i) polyclonal antibody

Polyclonal antibody normally obtains by relevant antigen of repeatedly subcutaneous (sc) in animal body or peritoneal injection and adjuvant.With difunctional or derivative reagent (as, sulfosuccinic imide maleimide aminobenzoyl ester (by the cysteine residues coupling reaction), N-hydroxyl succinimide (passing through lysine residue), glutaraldehyde, succinic anhydride, SOCl2 or R1N=C=NR (wherein R is different alkyl with R1)) make related antigen with treat immunity plant the immunogenic protein of apoplexy due to endogenous wind (as, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor) to carry out coupling be favourable.

Animal immune inoculation is combined acquisition with antigen, immunogenic conjugate or peptide or conjugate (respectively at rabbit type or Mus source type) by 1mg or 1ug with the Freund Freund's complete adjuvant of 3 times of volumes derivant.After 1 month animal many places subcutaneous injection is gone in the Freund Freund's complete adjuvant 1/5 to 1/10 booster immunization of original peptide or conjugate amount.After 7 to 14 days animal is drawn blood the antibody in the titration serum.The animal booster immunization is put down item until titre.Preferably, animal can be used same antigen, but comes booster immunization with different proteins and/or by the link coupled conjugate of different cross-linking agent.Conjugate can prepare as the protein blend compound in reconstitution cell is cultivated.Equally, flocculating agent such as Alumen also are applicable to the raising immunne response.

(ii) monoclonal antibody

Monoclonal antibody can be from the antibody population acquisition of homogeneous basically, and each antibody that promptly is included in this colony is identical except that a small amount of contingent natural mutation.Therefore, qualifier " monoclonal " refers to the mixture that the antibody characteristic is not an antibody specific.

For example, monoclonal antibody can use the hybridoma method that is at first proposed by people such as Kohler (Nature, 256:495 (1975)) to make, or makes with recombinant DNA method (U.S. Patent No. 4,816,567).

In hybridoma method, Mus or other suitable hosts animal, as hamster, available above-mentioned method immunity inoculation maybe can produce and is used for the bonded antibody of immunoreactive protein specific to cause that lymphocyte produces.Perhaps, lymphocyte can be in external immunity.Use suitable fusion agent (as Polyethylene Glycol) that lymphocyte and myeloma cell are merged then and generate hybridoma (Goding, MonoclonalAnibodies:Principle and Practice, pp.59-103 (Academic Press)).

The hybridoma of Huo Deing is inoculated in the proper culture medium and is grown like this, and culture medium preferably contains one or more and suppresses parental generation myeloma cell's growth of not merging or the material of surviving.For example, if parental generation myeloma cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), so usually, the used culture medium of hybridoma contains hypoxanthine, aminopterin and thymidine (HAT culture medium), can suppress the growth of HGPRT defective cell like this.

Preferable myeloma cell is that those fully merge, can produce antibody and to the cell as HAT culture medium sensitivity in antibodies selective produces cell highly stablely.In them, preferable myeloma cell line is a Mus source type myeloma cell line, as from MOPC-21 and MPC-11 Mus tumor (obtaining from California, USA Santiago Salk Institute Cell Distribution Center) and the cell of deriving and obtaining from SP-2 cell (obtaining from the American Type Culture Colletion of Maryland, USA Rochville).People source type myeloma and Mus-people's allos myeloma cell line is also described (Kozbor, J.Immunol., 133:3001 (1984) to some extent in the generation of human monoclonal antibodies; People such as Brodeur, Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

The culture medium that mensuration is used for the hybridoma growth produces at antigenic monoclonal antibody.Preferably, the binding specificity of the monoclonal antibody that hybridoma produces is measured as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) with immuno-precipitation or external binding assay.

The binding affinity of monoclonal antibody can be with measuring as Scatchard analytic process people such as (, Anal.Biochem., 107:220 (1980)) Munson.

After identifying the hybridoma that produces required specificity, affinity and/or active antibody, can come the sub-clone clone and make its growth (Goding by the limiting dilution step with standard method, Monoclonal Antibodies:Principlse and Practice, PP.59-103 (Academic Press, 1986)).The proper culture medium that is used for this purpose comprises, for example, and D-MEM or RPMI-1640 culture medium.Hybridoma can be grown as ascites tumour in animal body.

This sub-clone is that excretory monoclonal antibody can come appropriate separation to obtain as a-protein agarose gel, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatograph with routine immunization globulin purification step from culture medium, ascites liquid or serum.

The DNA of coding monoclonal antibody is easy to separate to obtain with conventional method and order-checking (as with can with the heavy chain of coding Mus source type antibody and the bonded oligonucleotide probe of gene specific of light chain).Hybridoma is as one of this DNA preferable source.In case after separating, DNA can be placed in the expression vector, then carrier is transfected into host cell such as Bacillus coli cells system, monkey COS cell line, Chinese hamster ovary (CHO) cell line or does not produce in addition among the myeloma cell of immunoglobulin, so that monoclonal antibody is synthetic in recombinant host cell.Recombinant expressed document about the DNA of encoding antibody in antibacterial has people such as Skerra at Curr.Opinion in Immunol., and 5:256-262 (1993) and Pluckthun be at Immunol.Revs., the summary among the 130:151-188 (1992).

In another example, antibody can separate acquisition with the technology that people such as McCafferty (Nature, 348:552-554 (1990)) describe from the antibody phage library.People (J.Mol.Biol., 222:581-597 (1991)) such as people such as Clackson (Nature, 352:624-628 (1991)) and Marks have described the method for separating Mus source type and people source type antibody with phage library respectively.After this, document discloses with chain reorganization (people such as Marks, Bio/Technology, 10:779-783 (1992)) the people source type antibody of generation high-affinity (in the nM scope), and with combination infect and body in recombinate and make up very big phage library (people such as Waterhouse, Nuc.Acids.Res., 21:2265-2266 (1993)).Therefore, these technology are promising methods different with the monoclonal antibody hybridoma technology of separating monoclonal antibody of routine.

DNA can also be by coded sequence (U.S. Patent No. 4,816,567 with homology Mus source type sequence replacing people source type heavy chain and constant region of light chain; People such as Morrison, Proc.Natl Acad.Sci.USA, 81:6851 (1984)) or modify by the sequence and the immunoglobulin coding sequence covalent bond of all or part coding NIg polypeptide.

Usually, this NIg polypeptide is used for substituting the constant region of antibody, or on the alternative antibody with the variable region in the bonded site of antigen to be formed with a chimeric bivalent antibody with the bonded antigen binding site of antigenic specificity and another and the different bonded antigen binding site of antigenic specificity.

The antibody of chimeric or heterozygosis also in the available protein synthetic chemistry known method (comprising those methods) with cross-linking agent in external preparation.For example, can make up immunotoxin by disulfide exchange reaction or formation thioether bond.Suitable reagent example comprises imino group mercaptides and methyl-4-sulfydryl butyryl polyurethane to be used for this purpose.

(iii) humanized and people source type antibody (human antibody)

The humanization of inhuman source type antibody is that this field is known.Usually, one or more amino acid residues in inhuman source have been introduced in the humanized antibody.These inhuman source type amino acid residue so-called " input " residues, they normally take out from " input " variable region.Humanization can be basically according to Winter and colleague's thereof method (people such as Jones, Nature, 321:522-525 (1986); People such as Reichman, Nature, 332:323-327 (1988); People such as Verhoeyen, Science, 239:1534-1536 (1988)) carry out, replace the corresponding sequence of people's antibody with Mus CDR or CDR sequence.Therefore, this " humanization " antibody is chimeric antibody (U.S. Patent No. 4,816567), wherein basically less than complete type variable region, people source by the corresponding sequence replacing of inhuman source type.In practice, humanized antibody is people source type antibody normally, and wherein, number of C DR residue and FR residue are substituted by the residue of the same loci of murine antibody.

The heavy chain and the type variable region, light chain people source that are used to produce humanized antibody are extremely important for reducing antigenicity.In so-called " only " method, the variable region sequences of murine antibody screens at all known people source type variable region sequences.People source type sequence that then will be the most close with the Mus type is as the humanization framework (FR) in the humanized antibody (people such as Sims, J.Immunol., 151:2296 (1993); People .J.Mol.Biol. such as Chothia, 196:901 (1992)).Another kind method has adopted the specific frame that obtains from the consensus sequence of the specific subclass of the light chain of everyone source type antibody or heavy chain.Identical framework can be used for several different humanized antibodies (people such as Carter, Proc.Natl.Acad.Sci.USA, 89:4285 (1992); People such as Presta, J.Immnol., 151:2623 (1993)).

It also is very important still having antigenic high affinity and other favourable biological property behind the antibody humanization.For realizing this purpose, according to preferred methods, humanized antibody is by analyzing the parental generation sequence and preparing with the method for the various humanization products of the threedimensional model generation of parental generation and humanization sequence.The threedimensional model of immunoglobulin normally can obtain, and is that these those skilled in the art are known.Computer program can be used to describe and show the three-dimensional conformation structure that selected immunoglobulin sequences is possible.Can analyze residue possibility role in selected immunoglobulin sequences by observing these demonstrations, promptly analyze the influence of residue for immunoglobulin to be selected and its antigen binding capacity.Like this, can from receptor and list entries, select the FR residue, obtaining required antibody character, as raising to the affinity of target antigen.Usually the CDR residue pair be combined with directly with antigen, very big influence.

Perhaps, can produce transgenic animal (as Mus) now, it can produce all compositions that produce people source type antibody under the condition that lacks at endogenous immunoglobulin when immunity inoculation.For example, there is description to say chimeric and heavy chain of antibody land (the J germ line mutation Mus _H) gene will suppress the generation of endogenous antibody fully.People's racial immunity globulin gene transfer is arranged in the antibody that will cause its generation people source when having antigen to attack in this germ line mutation Mus.See as people such as Jakobovits Proc.Natl.Acad.Sci.USA, 90:2551 (1993); People such as Jakobovits, Nature, 362:255-258 (1993); People such as Bruggermann, Year in Immuno., 7:33 (1993).Human antibody also can obtain (people such as Hoogenboom, J.Mol.Biol., 277:381 (1991) from phage display library; People such as Marks, J.Mol.Biol., 222:581-597 (1991)).

(iv) bi-specific antibody

Antibody bi-specific antibody (BsAbs) but be specificity in conjunction with the antibody of at least two kinds of different epitopes.This antibody can obtain from full length antibody or antibody fragment (as F (ab ') ₂Bi-specific antibody).

The method for preparing bi-specific antibody is that this field is known.The conventional production method of total length bi-specific antibody is according to two co expression that heavy chain immunoglobulin-light chain is right, and the specificity of two chains different (people such as Millstein, Nature, 305:537-539 (1983)).Because the heavy chain of immunoglobulin and the random assortment of light chain, these limbs hybridomas (quadroma) have produced the mixture that 10 kinds of different antibodies molecules are arranged, and wherein have only a kind of correct bispecific structure that has.Purification (using the affinity chromatography step usually) for correct molecule is quite to bother, and the product amount is very low.Same step at EMBO J., has disclosed people such as WO93/08829 and Traunecker among the 10:3655-3659 (1991).

According to another kind of method, the antibody variable region of required specificity (antibody-antigen binding site) and constant region for immunoglobulin sequence are merged.Best and immunoglobulin heavy chain constant region merges, and constant region comprises to small part hinge region, CH2 and CH3 district.Be preferably at least one fusions, CH (CH1) contains light chain in conjunction with required site.To the encode DNA of heavy chain immunoglobulin (can be the light chain of immunoglobulin if desired) fusions inserts in the independent expression vector, and cotransfection is gone in the suitable hosts organism then.Make up when optimum yield is provided when the polypeptide chain with three kinds of different proportions in example, this method can be regulated the intersegmental ratio of three peptide species sheets neatly.Yet, when at least two peptide species can cause high yield with the same ratio expression, or when ratio does not influence especially, the coded sequence of two or all three kinds polypeptide chain can be inserted in the expression vector.

In a preferable example of this method, bi-specific antibody is made up of (second binding specificity is provided on another arm) the heavy chain immunoglobulin (in the one arm first binding specificity being arranged) of hybridization and the heavy chain immunoglobulin-light chain of hybridization.Find that also this dissymmetrical structure makes that required bispecific chemical compound is easier and separates from unwanted immunoglobulin chain compositions, because the light chain immunoglobulin that exists only in half bispecific molecule provides easy method for separation.This method is open in the WO94/04690 that announced on March 3rd, 1994.The more detailed details that produces bi-specific antibody for example sees, people such as Suresh are at Methods in Enzymology, the description among the 121:210 (1986).

Bi-specific antibody comprises antibody crosslinked or " allos coupling ".For example, one of antibody in the allos conjugate can be coupled with Avidin, and another and biotin are coupled.This antibody for example be designed to make immune system cell targeting unwanted cells (U.S. Patent No. 4,676,980) and treatment HIV infect (WO91/00360, WO92/200373).Allos conjugate antibody can make with any cross-linking method easily.Suitable crosslinking agent is that this field is known, and in U.S. Patent No. 4,676, discloses many crosslinking technologicals in 980.

The technology that produces bi-specific antibody from antibody fragment is also described in the literature to some extent.That following technology also can be used for producing bivalence but needn't be the antibody fragment of bispecific.For example, the Fab ' fragment that obtains from escherichia coli can form bivalent antibody in external chemical bond.See people such as Shalaby, J.Exp.Med., 175:217-225 (1992).

Directly from reconstitution cell is cultivated, prepare and separate the segmental multiple technologies of bivalent antibody and have description.For example, leucine zipper can produce bivalence heterodimer (people such as Kostelny, J.Immunol., 148 (5): 1547-1553 (1992)).Be connected with the Fab ' part of two different antibodies by gene fusion from the peptide section protein of the leucine zipper of Fos to Jun.Be reduced into monomer at the antibody homodimer of hinge region and be oxidized to the antibody heterodimer then again." double antibody " technology (Proc.Natl.Acad.Sci.USA, 90:6444-6448 (19993)) that people such as Hollinger describe provides another kind of preparation bispecific/bivalent antibody segmental thinking.Comprise variable region of heavy chain (V in the fragment _H), it is by very short joint and variable region of light chain (V _LThereby) connect two interregional can not pairings making on the same chain.Therefore the V on fragment _HAnd V _LThe district be forced to another fragment on complementary V _LAnd V _HTherefore district's fragment pairing has formed two antigen binding sites.Also reported in addition with strand Fv (sFv) the preparation segmental thinking of bispecific/bivalent antibody (seeing people such as Gruber, J.Immunol., 152:5368 (1994)).

B. the preparation of lyophilized formulations

As above-mentioned prepared protein of interest matter after, can make " lyophilizing before preparation ".Protein content in the preparation before the lyophilizing can wait to determine according to required dosage volume, administering mode.When selected protein was complete antibody (as anti-IgE or Anti-HER 2), typical initiation protein concentration should be about 2mg/mL to about 50mg/mL, the preferable extremely about 40mg/mL of 5mg/mL that is about, and the best is about 20-30mg/mL.Protein exists in solution usually.For example, protein can be about in the pH buffer of 4-8 at pH, and pH the best is about 5-7.Typical buffer comprises histidine, phosphate, Tris, citrate, succinate and other organic acid buffer.Buffer concentration is about 1mM to about 20mM, or about 3mM about 15mM extremely, and this is that basis is determined as buffer and required preparation (as reconstituted formulation) isotonicity.Preferable buffer is a histidine, proves that below it has frozen-dried protective (lyoprotective) characteristic.The succinate buffer also is a kind of useful buffer in addition.

Freeze drying protectant is added in the preparation before the lyophilizing, and in preferred embodiment, freeze drying protectant is nonreducing sugar class such as sucrose or trehalose.The amount of the freeze drying protectant in the preparation before the lyophilizing should make the preparation that obtains when rebuilding be isotonicity.Yet the reconstituted formulation that height oozes also is suitable for.In addition, the amount of freeze drying protectant also should not be hanged down excessively and cause protein degradation/cohesion that unsatisfactory amount takes place when lyophilizing.When freeze drying protectant is saccharide (as sucrose or trehalose) and protein when being antibody, in the preparation before the lyophilizing concentration of typical freeze drying protectant for about 10mM to about 400mM, preferable be about 30mM about 300mM extremely, preferably about 50mM is about 100mM extremely.

The combination of each protein and freeze drying protectant all will be selected the ratio of protein and freeze drying protectant.When selected protein is that antibody and freeze drying protectant are when being saccharide (as sucrose or trehalose) with the isotonicity reconstituted formulation that generates high concentration protein; the ratio of freeze drying protectant and antibody be about 100 to about 1500 moles of freeze drying protectants than 1 mole of antibody; be preferably about 200 to about 1000 moles of freeze drying protectants than 1 mole of antibody, for example about 100 to about 600 moles (be more preferred from about 200 to about 600 moles freeze drying protectant than 1 mole of antibody.

In preferred embodiment of the present invention, discovery need add a kind of surfactant in the preparation before lyophilizing.Perhaps, in lyophilized formulations and/or reconstituted formulation, also need to add surfactant.Exemplary surfactants comprises nonionic surfactant such as polysorbate (as polysorbate20 or 80); Poloxamer (poloxamer) (as poloxamer 188); Triton; Sodium lauryl sulphate (SDS); Sodium laurylsulfate; Octyl group glucosides sodium; Lauryl, myristyl, inferior oil base or octadecyl sulfobetaines; Lauryl, myristyl, inferior oil base or octadecyl sarcosine; Inferior oil base, myristyl or cetyl betanin; Dodecanamide propyl, cocamidopropyl propyl amide, inferior oleamide propyl group, myristamide propyl group, palmitic amide propyl group or isooctadecane base amido propyl betaine (as dodecanamide propyl); Myristamide propyl group, palmitic amide propyl group or isooctadecane base amido propyl dimethylamine; SODIUM METHYL COCOYL TAURATE or methyl oleyl taurine disodium; And MONAQUAT ^TMThe copolymer (as Pluronics, PF68 etc.) of series (MonaIndustries, Inc., Paterson, New Jersey), Polyethylene Glycol, polypropylene glycol and ethylene glycol and propylene glycol.Protein condenses reduced and makes the granulating trend minimum of reconstruction back preparation when the addition of surfactant should make reconstruction.For example, the amount of the surfactant in the preparation before the lyophilizing is about 0.001-0.5%, and that preferable is about 0.005-0.05%.

In some embodiments of the invention, the mixture of freeze drying protectant (as sucrose or trehalose) and filler (as mannitol or glycine) can be used for the preparation of the preceding preparation of lyophilizing.Filler can generate the lyophilized cake of homogeneous and not have wherein too much perforate.

Can comprise other pharmaceutically acceptable carrier, excipient or stabilizing agent in the preparation (and/or lyophilized formulations and/or reconstituted formulation) before the lyophilizing, as Remington ' s Pharmaceutical Sciences (16thedition, Osol, A.Ed. (1980)) middle description, as long as they do not have negative effect to the required character of preparation.Dosage and concentration that acceptable carrier, excipient or stabilizing agent adopt should be nontoxic to receptor, and they comprise other buffer, antiseptic, cosolvent, antioxidant such as ascorbic acid and methionine, chelating agen such as EDTA, metal complex for example Zn-protein coordination compound, biodegradable polymer such as polyester and/or salify counter ion such as sodium.

The preparation here also can contain multiple proteins to be used for treating especially indication, and preferably these have the protein of complementary activity that other protein is not had negative effect.For example, in a preparation, can provide two or more and HER2 receptor or the bonded antibody of IgE.And the antibody of anti-HER2 and anti-VEGF can be used in combination in same preparation.These protein can effectively be measured the purpose that is intended to reach and be present in the described combination.

The preparation that is used for vivo medicine-feeding must be aseptic.This can by in lyophilizing and before rebuilding or the filtration of flowing through aseptic filter membrane afterwards realize.Perhaps, whole mixture can be by for example realizing aseptic in about 30 minutes to the outer component of isolating protein at about 120 ℃ of autoclavings.

After protein, freeze drying protectant and other component mixing arbitrarily, but lyophilized formulations.Realize that this purpose can adopt different freezer dryers, as Hull50 ^TM(Hull, USA) or GT20 ^TM(Leybold-Heraeus, Germany) type freezer dryer.Lyophilization is earlier that preparation is freezing, is being suitable for making refrigerated ice distillation under the elementary exsiccant temperature then.Under this condition, the product temperature is lower than the eutectic point or the disintegrate point of preparation.Usually elementary exsiccant shelf temperature is-30 ℃ to 25 ℃ (product should keep frozen state between elementary dry period) approximately, and suitable pressure is about 50 to 250mTorr.Dry required time mainly determines that by preparation, the size of container (as vial) of placing sample and the volume of type and liquid the time can be in several hours to several days (for example 40-60 hour).The secondary drying step can be carried out at about 0-40 ℃, and this mainly is according to the type and size of used container and proteinic kind.Yet, find that here the secondary drying step is inessential.For example, the shelf temperature removed of the whole water of lyophilized products is about 15-30 ℃ (20 ℃ according to appointment).Time that secondary drying is required and pressure should be able to generate suitable lyophilized cake, they and for example temperature and other relating to parameters.The secondary drying time determined by required residual moisture level in the product, usually need be at least about 5 hours (as 10-15 hour).Pressure can be identical with used pressure in the elementary drying steps.The lyophilization condition can according to preparation and the bottle size and different.

In some instances, wish lyophilized protein formulation in container, and can carry out proteinic reconstruction therein to avoid transfer step.Container in this example for example can be 3,5,10,20,50 or the bottle of 100cc.

Usually, lyophilizing should make moisture in the lyophilized formulations less than about 5%, and is preferable less than about 3%.

C. the reconstruction of lyophilized formulations

Usually when needs during to patient's administration, required step is to rebuild lyophilized formulations with diluent, make the protein concentration in the reconstituted formulation be at least 50mg/mL, for example be that about 50mg/mL is to about 400mg/mL, be more preferred from about 80mg/mL to about 300mg/mL, best is that about 90mg/mL is to about 150mg/mL.When needs subcutaneous defeated when passing reconstituted formulation, in the reconstituted formulation so high protein concentration be considered to useful especially.Yet,,, need the protein concentration low slightly (for example the protein concentration in the reconstituted formulation is about 5-50mg/mL, or is about 10-40mg/mL) in the reconstituted formulation as intravenous administration for other route of administration.In some instances, high many of the concentration in the preparation of the protein concentration in the reconstituted formulation before than lyophilizing.For example, in the reconstituted formulation protein concentration be the preparation before the lyophilizing about 2-40 doubly, be preferably 3-10 doubly, the best is 3-6 times (as at least 3 times or at least 4 times).

Rebuild and normally to carry out, guaranteeing complete hydration, but also can adopt other temperature as required at about 25 ℃.The time of rebuilding is relevant with proteinic amount with kind, the excipient of for example diluent.Typical diluent comprises sterilized water, injection antibacterial (BWFI), pH buffer (for example phosphate buffer), aseptic salt solution, Ringer's solution or Glucose Liquid.Diluent can also contain antiseptic, typical preservatives as mentioned above, preferably aromatic alcohol such as phenyl or phenols alcohol.The consumption of antiseptic decides with the proteinic compatibility and antiseptic effect by measuring under the different concentration of preservatives.For example, if antiseptic is aromatic alcohol (as a benzyl alcohol), its content can be about 0.1-2.0%, is preferably about 0.5-1.5%, but the best is about 1.0-1.2%.

Preferably, particle diameter should be less than 6000 more than or equal to the granule of 10 μ m in each bottle reconstituted formulation.

D. the administration of reconstituted formulation

Reconstituted formulation can be come mammal to treating with protein with known method, preferably people's administration, method such as intravenous administration such as intravenous injection or the continuous infusion in a period of time, by in the intramuscular, intraperitoneal, marrowbrain, in subcutaneous, the intraarticular, synovial cavity, in the sheath, in the oral cavity, skin surface or inhalation route administration.

In preferred embodiment, reconstituted formulation is come the mammal administration by subcutaneous (be skin below).For this purpose, preparation is used syringe and is injected.Yet also available other is used for the equipment of administration, as injection device (as Iniect-ease ^TMAnd Genject ^TMDevice); Injection pen is (as GenPen ^TM); Needleless device is (as MediJector ^TMAnd BioJector ^TM) and the subcutaneous delivery system.

Proteinic proper dosage (" treatment effective dose ") and disease, disease seriousness and process, protein for example to be treated are administered for still treatment of prevention, former treatment, patient's medical history and relevant to proteinic reaction, used kinds of protein and attending doctor's processing.Protein can be once or in a series of courses of treatment to patient's administration, and can be according to the diagnosis administration at any time of front.Protein can be used for the treatment of separately or be used in combination with other medicine or therapy that can be used for treating disease.

When selected protein is antibody, no matter be one or many separate administration for example, be about 0.1-20mg/kg to the initial selected dosage of patient's administration.Yet, also available other dosage.The progress of this therapy is easy to monitor with routine techniques.

In the example of Anti-HER 2, it is the cancer of feature that the treatment effective dose of antibody should treat or prevent with the overexpression of HER2 receptor.Think that the reconstituted formulation of Anti-HER 2 can be used for treating breast carcinoma, ovarian cancer, gastric cancer, carcinoma of endometrium, salivary-gland carcinoma, pulmonary carcinoma, renal carcinoma, colon cancer and/or bladder cancer.For example, Anti-HER 2 can be used for treating ductal carcinoma in situ (DCIS).Typical Anti-HER 2 dosage is 1-10mg/kg in the one or many separate administration.

Anti-IgE preparation can be used for treating or preventing the allergic disease of IgE mediation, for example parasite infection, interstitial cystitis and asthma.The treatment effective dose of anti-IgE antibodies (for example about 1-15mg/kg) can come the administration to the patient according to the disease of for example treatment.

E. the product of Sheng Chaning

Another aspect of the present invention provides a kind of product and its reconstruction and/or explanation of usage that contains lyophilized formulations of the present invention.Product comprises container.Suitable containers comprises, for example vial, bottle (for example two chambers bottle), syringe (for example double-chamber syringe) and test tube.Container can make with various materials such as glass or plastics.Lyophilized formulations is arranged in the container, on the container or and container supporting label the method for rebuilding and/or using has been described.For example, label can be indicated lyophilized formulations is rebuild to above-mentioned protein concentration.Label can illustrate that also preparation is applicable to subcutaneous injection.The container that preparation is housed can be muptiple-use use bottle, and it can repeat the administration (as administration 2-6 time) of reconstituted formulation.Product also can comprise the container that another has suitable diluents (as BWFI).When mixed diluent and lyophilized formulations, the protein ultimate density in the reconstituted formulation is at least 50mg/mL.From commercial and user perspective, product also can comprise other article, comprises other buffer, diluent, filter membrane, syringe needle, syringe and the package insert that has operation instruction.

Can fully understand the present invention with reference to following example.Yet they should not be construed as limiting the scope of the invention.All documents are all quoted reference as proof.

Embodiment 1

Anti-HER2 preparation

HER2 proto-oncogene product (p185 ^HER2) overexpression relevant with various invasion human malignant lesions.The Mus type monoclonal antibody that is called muMAb4D5 can directly act on p185 ^HER2Extracellular region territory (ECD).The muMAb4D5 molecule by humanization to attempt the immunogenicity by reducing it and to make its backer's bulk effect device function improve clinical effectiveness (seeing WO 92/22653).This example has been described and has been developed the lyophilized formulations that comprises the total length humanized antibody huMAb4D5-8 that describes among the WO 92/22653.

When the exploitation lyophilized formulations, at first screen excipient and buffer by measuring the stability of protein after lyophilizing and reconstruction.Freeze-dried protein in each preparation is made further stability study to measure the stability of protein in its storage life.These further researchs are normally carried out under the above-mentioned storage conditions temperature that provides, then according to Arrhenius kinetics with the required activation energy of data estimation degradation reaction (people such as Cleland, Critical Reviews in Therapeutic DrugCarrier System 10 (4): 307-377 (1993)).Calculate the storage life of the protein formulation of under the storage conditions that provides, estimating then with activation energy.

In the screening study, incubation was studied some freeze dried recombinant humanized Anti-HER 2 (rhuMAbHER2) stability of formulation afterwards under 5 ℃ (storage conditions that provide) and 40 ℃ (further condition of stability study) in early days.When liquid state, discovery rhuMAB HER2 degrades by desamidation (30 agedoites of light chain) and forms different aspartic acid (102 aspartic acids of heavy chain) by cyclic imide intermedium butanimide.Minimum in the reaction of pH5.0 desamidation, cause degraded to be essentially butanimide.When pH6.0, in the liquid protein preparation, find to have the desamidation reaction of higher amount.Therefore, at (a) 5 or 10mM succinate buffer, pH5.0 and (b) 5 or the 10mM histidine buffering liquid, pH6.0 is the research lyophilized formulations down.Buffer all contains surfactant polysorbate20 (polysorbas20 ^TM), it can be used for reducing the proteinic cohesion trend of reconstruction and makes reconstruction back granule form minimum.These buffer can not use with various saccharides with reaching.With protein with 5.0,21.0 or the concentration of 25.0mg/mL allocate buffer into.Lyophilized formulations is then measured proteinic stability after 5 ℃ and 40 ℃ of 2 weeks of preservation.In freezer dryer, bottle under about-55 ℃ shelf temperature freezing about 5 hours, then 5 ℃ of shelf temperature, under the 150mTorr pressure elementary dry 30 hours, and with 20 ℃ shelf temperature secondary drying 10 hours to be dried to the residual moisture of 1-2%.The main degradation pathway of this protein when lyophilizing is cohesion, and therefore the yield of the complete non-denatured protein matter of available non-degeneration size exclusion chromatographic determination is measured protein stability (% of whole protein sees the following form 2).

At the 10mM sodium succinate, measure of the influence of various freeze drying protectant saccharides among the pH5.0 (table 2) for the stability of freeze-dried protein.Under high glucose concentration (250-275mM) and low-protein concentration (5.0mg/mL), trehalose and lactose can make stable and prevent its cohesion at the freeze-dried protein in 40 ℃ of 2 weeks of preservation.Yet lactose is a kind of reducing sugar, find it when 40 ℃ of long-term preservations will with proteins react.The proteic preparation of 5.0mg/mL that contains Sorbitol or mannitol will produce protein condenses after 40 ℃ of 2 weeks of preservation.Under increased protein concentration (21.0mg/mL), after lyophilizing preservation under two kinds of conditions, contain the protein of cohesion by the preparation of the combination of mannitol or mannitol and Sorbitol or glycine.On the contrary, trehalose and sucrose can prevent cohesion under two kinds of storage conditions.

Measure the long-time stability of the preparation that contains 250mM trehalose and 250mM lactose now.40 ℃ of preservations 9 months or 5 ℃ of preservations after 12 months, the percentage ratio that contains whole protein in the preparation of trehalose does not change.And, keep constant (with original the same) after 3 months or at the percentage ratio of 25 ℃ of preservations whole protein after 6 months 40 ℃ of preservations for the lactose preparation.Contain the preparation of trehalose can be under the room temperature of control (15-30 ℃) down preservation 2 years and the percentage ratio of whole protein is not significant changes.

After 40 ℃ of 2 weeks of preservation, pH6.0 is arranged, the histidine of 10mM and the preparation of mannitol ratio have pH5.0, and the succinate of 10mM and the preparation of mannitol contain cohesion protein still less.This possibility of result is because the stablizing effect of single histidine contribution.Yet it is after 40 ℃ of 2 weeks of preservation, single with in histidine or the preparation significant cohesion being arranged with histidine/mannitol.The sucrose that adds the amount (10mg/mL) that equates with mannitol in the histidine preparation can prevent under two kinds of storage conditions that cohesion from making protein stabilization.Do not improve proteinic stability with glycine and mannitol, the stability that sucrose/glycine preparation then provides with sucrose/the mannitol preparation is identical.This result shows that further sucrose helps to prevent the cohesion of freeze-dried protein when preservation.

Table 2

Component before the lyophilizing		The percentage ratio of whole protein ^a
Component before the lyophilizing		The percentage ratio of whole protein ^a			Protein ^b(mg/mL)	Prescription	Liquid (5 ℃)	Lyophilizing (2 weeks, 5 ℃)	Lyophilizing (2 weeks, 40 ℃)
	The 10mM sodium succinate, pH5.0				Protein ^b(mg/mL)	Prescription	Liquid (5 ℃)	Lyophilizing (2 weeks, 5 ℃)	Lyophilizing (2 weeks, 40 ℃)
	The 10mM sodium succinate, pH5.0				5.0	The 275mM trehalose, 0.01% polysorbas20 ^TM	98.9	99.1	98.9
5.0	The 275mM lactose, 0.01% polysorbas20 ^TM	96.8	96.5	96.6	5.0	The 275mM trehalose, 0.01% polysorbas20 ^TM	98.9	99.1	98.9
5.0	The 275mM lactose, 0.01% polysorbas20 ^TM	96.8	96.5	96.6	5.0	The 275mM Sorbitol, 0.01% polysorbas20 ^TM	99.4	99.3	95.4
5.0	The 250mM mannitol, 0.01% polysorbas20 ^TM	100.0	99.9	98.8	5.0	The 275mM Sorbitol, 0.01% polysorbas20 ^TM	99.4	99.3	95.4
5.0	The 250mM mannitol, 0.01% polysorbas20 ^TM	100.0	99.9	98.8	5.0	The 250mM trehalose, 0.01% polysorbas20 ^TM	100.0	99.9	100.0
5.0	The 250mM lactose, 0.01% polysorbas20 ^TM	100.0	100.0	100.0	5.0	The 250mM trehalose, 0.01% polysorbas20 ^TM	100.0	99.9	100.0
5.0	The 250mM lactose, 0.01% polysorbas20 ^TM	100.0	100.0	100.0	21.0	The 250mM trehalose, 0.2% polysorbas20 ^TM	99.3	99.1	99.1
21.0	250mM sucrose, 0.2% polysorbas20 ^TM	99.6	99.6	99.7	21.0	The 250mM trehalose, 0.2% polysorbas20 ^TM	99.3	99.1	99.1
21.0	250mM sucrose, 0.2% polysorbas20 ^TM	99.6	99.6	99.7	21.0	The 250mM mannitol, 0.01% polysorbas20 ^TM	100.0	94.6	94.0
21.0	188mM mannitol/63mM Sorbitol,	99.8	98.6	96.5	21.0	The 250mM mannitol, 0.01% polysorbas20 ^TM	100.0	94.6	94.0

	0.01% polysorbas20 ^TM
	0.01% polysorbas20 ^TM				21.0	250mM mannitol/25mM glycine, 0.01% polysorbas20 ^TM	99.5	96.5	96.4
	The 10mM histidine, pH6.0				21.0	250mM mannitol/25mM glycine, 0.01% polysorbas20 ^TM	99.5	96.5	96.4
	The 10mM histidine, pH6.0				21.0	There is not sugar, 0.01% polysorbas20 ^TM	100.0	99.9	98.9
21.0	54.9mM mannitol, 0.01% polysorbas20 ^TM	100.0	99.9	99.2	21.0	There is not sugar, 0.01% polysorbas20 ^TM	100.0	99.9	98.9
21.0	54.9mM mannitol, 0.01% polysorbas20 ^TM	100.0	99.9	99.2	21.0	29.2mM sucrose/266.4mM glycine, 0.01% polysorbas20 ^TM	100.0	100.0	99.6
21.0	54.9mM mannitol/266.4mM glycine, 0.01% polysorbas20 ^TM	100.0	99.8	98.9	21.0	29.2mM sucrose/266.4mM glycine, 0.01% polysorbas20 ^TM	100.0	100.0	99.6
21.0	54.9mM mannitol/266.4mM glycine, 0.01% polysorbas20 ^TM	100.0	99.8	98.9	21.0	54.9mM mannitol/29.2mM sucrose, 0.01% polysorbas20 ^TM	99.8	100.0	99.7

A. the component of whole protein with the peak area of non-degeneration size exclusion HPLC and non-denatured protein matter with respect to the value of the total peak area that comprises condensation product measure (TSK3000 SW XL post, TosoHaas, flow velocity are 1.0mL/min; Use the phosphate buffer eluting; 214 and 280nm under detect).Protein formulation before lyophilizing after (liquid, 5 ℃), the lyophilizing and 5 ℃ or 40 ℃ 2 week of preservation post analysis.

B. containing the proteinic preparation of 5mg/mL rebuilds (20mL, 5.0mg/mL protein) with distilled water and contains the proteinic preparation of 21mg/mL injection antibacterial (BWFI, 0.9% benzyl alcohol; 20mL, 20mg/mL protein) rebuild.

Because the volume restrictions of subcutaneous administration (≤1.5mL) and the dosage demand (〉=100mg), therefore need the defeated of increased protein concentration to pass usually.Yet, because protein has the trend of cohesion in processing under high concentration, to its intractable (for example extracting out) and aseptic filtration, so increased protein concentration (〉=50mg/mL) be difficult to usually realize in process of production.Freeze-drying process provides a kind of method that allows protein to concentrate in addition.For example, protein is packed in the bottle into lyophilizing then with certain volume (Vf).Then freeze-dried protein is rebuild with the water or the antiseptic (as BWFI) of the little volume (Vr) (as Vr=0.25Vf) of original volume, making to rebuild in the solution has higher protein concentration.This method also makes buffer and excipient concentrate.For subcutaneous administration, wish that solution is isoosmotic.

Trehalose amount among the minimizing lyophilizing rhuMAB HER2 is to form isosmotic solution when being reconstructed into 100mg/mL protein.Be determined at pH5.0,5mM sodium succinate and pH6.0, the stablizing effect (table 3) that trehalose becomes with concentration under 5mM histidine, the 25.0mg/mL protein.Between trehalose concentration was from 60 to 200mM, freeze-dried protein was in the not significant cohesion after 4 weeks of 40 ℃ of incubations.These preparations are rebuild in 20mL injection antibacterial (BWFI, USP, 0.9% benzyl alcohol).50mM trehalose preparation (5mM sodium succinate), is rebuild in 4mL BWFI (100mg/mL protein) after 4 weeks at 40 ℃ of incubations, and the preparation of generation has the cohesion of recruitment a little.The reconstituted formulation of preservation provides and can repeatedly take out and the advantage of not microbiological contamination from same bottle.When using Inoculating needle, the preparation in this bottle just can divide taking-up several times.

Table 3

Component before the lyophilizing		The percentage ratio of whole protein ^a
Component before the lyophilizing		The percentage ratio of whole protein ^a			Protein (mg/mL)	Prescription	Liquid (5 ℃)	Lyophilizing (4 weeks, 5 ℃)	Lyophilizing (4 weeks, 40 ℃)
	The 5mM sodium succinate, pH5.0				Protein (mg/mL)	Prescription	Liquid (5 ℃)	Lyophilizing (4 weeks, 5 ℃)	Lyophilizing (4 weeks, 40 ℃)
	The 5mM sodium succinate, pH5.0				25.0	The 50mM trehalose, 0.01% polysorbas20 ^TMb	100.0	100.0	99.5
25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.9	25.0	The 50mM trehalose, 0.01% polysorbas20 ^TMb	100.0	100.0	99.5
25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.9	25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.2
25.0	The 100mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.7	25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.2
25.0	The 100mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.7	25.0	The 150mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.8
25.0	The 200mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	100.0	25.0	The 150mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.8
25.0	The 200mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	100.0		The 5mM histidine, pH6.0
25.0	38.4mM mannitol/20.4mM sucrose, 0.01% tween ^TM	100.0	100.0	99.3		The 5mM histidine, pH6.0
25.0	38.4mM mannitol/20.4mM sucrose, 0.01% tween ^TM	100.0	100.0	99.3	25.0	38.4mM mannitol/20.4mM sucrose, 0.01% tween ^TM c	100.0	100.0	99.4
25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM d	100.0	100.0	99.8	25.0	38.4mM mannitol/20.4mM sucrose, 0.01% tween ^TM c	100.0	100.0	99.4
25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM d	100.0	100.0	99.8	25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.4
25.0	The 100mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.6	25.0	The 60mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.4
25.0	The 100mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	99.6	25.0	The 150mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	100.0
25.0	The 200mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	100.0	25.0	The 150mM trehalose, 0.01% polysorbas20 ^TM	100.0	100.0	100.0

A. the component of whole protein is measured with non-degeneration size exclusion HPLC, and be defined as the peak area of non-denatured protein matter with respect to the value of the total peak area that comprises condensation product (TSK3000 SW XL post, TosoHaas, flow velocity are 1.0mL/min; Use the phosphate buffer eluting; 214 and 280nm under detect).Protein formulation before lyophilizing after (liquid, 5 ℃), the lyophilizing and 5 ℃ or 40 ℃ 4 week of preservation post analysis.Preparation injection antibacterial (BWFI, USP, 0.9%w/w benzyl alcohol; 20mL, 22mg/mL protein) rebuild.

B. using 4mL BWFI (0.9% benzyl alcohol) to be reconstructed into protein concentration is 100mg/mL.

C. using 4mL BWFI (1.1% benzyl alcohol) to be reconstructed into protein concentration is 100mg/mL.

D. sample is 5 ℃ or 40 ℃ of 2 weeks of incubation, and using 20mL BWFI (0.9% benzyl alcohol) to be reconstructed into protein concentration then is 22mg/mL.

Now, research rhuMAb HER2 is as the medicine of treatment breast carcinoma.Take 2mg/kg protein weekly for the patient.Because these patients' average weight is 65kg, therefore, on average dosage is 130mg rhuMAb HER2 weekly.For subcutaneous administration, volume injected for or be acceptable less than 1.5ml, therefore, the rhuMAb HER2 protein concentration of weekly subcutaneous administration is about 100mg/mL (130mg mean dose/1.5mL).As mentioned above, this increased protein concentration is difficult to generate and keeps stable.For reaching increased protein concentration, rhuMAb HER2 can be formulated into the protein concentration of 25mg/mL: (a) 5mM sodium succinate, pH5.0 or (b) 5mM histidine, pH6.0 and 60mM trehalose, 0.01% polysorbas20 ^TMIn and lyophilizing.During lyophilizing 18mL protein formulation branch packed in the 50cc bottle.In freezer dryer, bottle under about-55 ℃ shelf temperature freezing about 5 hours, then 5 ℃ of shelf temperature, under the 150mTorr pressure elementary dry 30 hours, and with 20 ℃ shelf temperature secondary drying 10 hours to be dried to the residual moisture of 1-2%.Place the thermocouple that contains placebo (nonprotein preparation) to show that the product of bottle maintains below-10 ℃ in whole elementary drying.Prevention in regular turn when lyophilizing studies show that at elementary dry back residual moisture usually less than 10%.

Then freeze-dried protein with 4 or 20mL BWFI (0.9 or 1.1% benzyl alcohol) rebuild to generate spissated protein solution:

(a) 4mL:102mg/mL rhuMAb HER2,245mM trehalose, 21mM sodium succinate, pH5.0 or 21mM histidine, pH6.0,0.04% polysorbas20 ^TM

(b) 20mL:22mg/mL rhuMAb HRR2,52mM trehalose, 4mM sodium succinate, pH5.0 or 4mM histidine, pH6.0,0.009% polysorbas20 ^TM

Lyophilized formulations is reconstructed into the protein concentration of 22mg/mL after 40 ℃ of 4 weeks of preservation, rise along with the proteinic cohesion amount of the minimizing of trehalose concentration demonstrates slightly.The stability of freeze-dried protein then is not subjected to the influence of reconstruct volume.As shown in Figure 1, freeze-dried protein (60mM trehalose, 5mM sodium succinate, pH5.0,0.01% polysorbas20 ^TM) behind 40 ℃ of incubations with 4 or the 20mL BWFI amount of rebuilding the back whole protein be identical.

The result of table 3 shows certain relation between trehalose concentration and protein stability.Be further to determine this relation, carried out incubation 91 days at 40 ℃ of sodium succinate or histidine preparations to the trehalose that contains different compound concentrations.Be determined at the stability that changes with trehalose and protein mol ratio under each trehalose concentration then.As shown in Figure 2, along with the decline of trehalose concentration, protein stability all significantly decreases in two kinds of preparations.Adopt two kinds of different buffer in these preparations, promptly succinate and histidine are not significantly distinguished, and this shows that stabilizing agent main under this condition is a trehalose.In addition, the minimizing of the whole protein of finding in two kinds of preparations even also be acceptable in its storage life for the low trehalose concentration preparation that is deposited in 2-8 ℃.Yet, control ambient-temp-stable (maximum temperature is 30 ℃) if desired, according to product stability standard (standard of preservation amount of remaining whole protein after 2 years), may need higher trehalose concentration (trehalose: the ratio of protein 〉=600:1).Usually, control room temperature storage conditions is 40 ℃ of stable preservations 6 months and in 30 ℃ of preservations equivalence in 2 years.

As shown in Figure 3,250mM trehalose preparation is 40 ℃ of preservations 6 months and no change, and the trehalose preparation of 60mM is then unstable.As fruit product in the standard of preserving the end of term be, for example with non-degeneration size exclusion chromatographic determination whole protein greater than 98% arranged, the preparation of 60mM trehalose concentration then needs freezing so.

In the screening study formerly, find that also sucrose can be after lyophilizing and prevent the cohesion of rhuMab HER2 during follow-up preservation.For after reconstruction, obtaining isosmotic solution, must significantly reduce sucrose concentration to be used for subcutaneous administration (formulation components and protein concentrate 4 times approximately).In screening study, the employing of the sucrose of equal in quality concentration and mannitol (filler) has prevented proteinic cohesion.Select the sucrose of low concentration to be used for the subcutaneous preparation of using of effective rhuMAb HER2 now with mannitol (identical mass concentration).Freeze-dried protein solution (25mg/mL protein, 5mM histidine, pH6.0,38.4mM (7mg/mL) mannitol, 20.4mM (7mg/mL) sucrose, 0.01% polysorbas20 ^TM), method therefor is identical with the preparation of 60mM trehalose, is elementary exsiccant cycle stretch-out to 54 hour.After 40 ℃ of 4 weeks of preservation, with 4.0 or 20.0mL BWFI rebuild back condensation product amount and slightly increase (table 3).The protein condenses amount with to being reconstructed into 22 or the coming to the same thing of 100mg/mL protein concentration (Fig. 4).Identical with 60mM trehalose preparation, mannitol/sucrose preparation will form less intact proteins quality at 40 ℃ of preservation certain hours.Sucrose is 120 to 1 with proteinic mol ratio in the said preparation, and this combination ratio that shows mannitol/sucrose under the stabilisation sugar condition of identical mol ratio is single with trehalose more effective (Fig. 2 and 4).

In the example formerly, measured lyophilizing rhuMAb HER2 stability of formulation with the variation of temperature situation.These studies have shown that the preparation of trehalose and mannitol/sucrose can prevent the protein degradation of lyophilizing attitude under high temperature (40 ℃).Yet the stability of protein after reconstruction and preservation is not determined in these tests.After rebuilding with BWFI, freeze dried rhuMAb HER2 preparation can be used for multiple dosing.Particularly, bottle capacity (450mg rheMAb HER2) but be designed to three patients of average out to medicament (each dosage be 130mg rhuMAb HER2) be provided.Because medicine takes weekly once, therefore, bottle must be after reconstruction at least three weeks of preservation, stable for determine that rhuMAb HER2 can keep after reconstruction, carry out stability study at 5 ℃ and 25 ℃ of rhuMAb HER2 preparations now to reconstruction.

For subcutaneous administration, it is 100mg/mL (4mL BWFI) that preparation can be reconstructed into protein concentration.Under this increased protein concentration, the protein ratio is reconstructed into the easier cohesion of intravenous formulation of 22mg/mL protein (20mL BWFI).Measure the cohesion situation (loss of whole protein) of four kinds of rhuMAb HER2 preparations in the first preceding example.As table 4 to shown in 6, be reconstructed into 22 and the stability of formulation of 100mg/mL protein concentration as broad as long.And these preparations can make whole complete 90 days of protein maintenance and can keep protein all complete 30 days at 25 ℃ at 5 ℃, but this shows reconstituted formulation freezing at least 90 days.Different with the freeze-dried protein stability in the first preceding example, trehalose concentration does not have influence (table 7) for protein stability in the said preparation.

Table 4

Protein concentration be the rhuMAb HER2 of 25mg/mL at the 5mM sodium succinate, pH5.0,60mM trehalose, 0.01% polysorbas20 ^TMThe stability of reconstituted formulation after the middle lyophilizing

Time (natural law)	The percentage ratio of whole protein
	The percentage ratio of whole protein				22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃	22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃	0	99.9	99.9	99.7	99.7
14	ND	100.0	ND	100.0	0	99.9	99.9	99.7	99.7
14	ND	100.0	ND	100.0	30	100.0	100.0	100.0	100.0
91	99.8	ND	100	ND	30	100.0	100.0	100.0	100.0

Sample is rebuild with 4.0mL or 20.0mL BWFI (1.1% or 0.9% benzyl alcohol), is deposited in 5 ℃ or 25 ℃ then.The percentage ratio of whole protein is defined as the component (fraction) of non-degeneration peak area with non-degeneration size exclusion chromatographic determination.ND is not for measuring.

Table 5

Protein concentration be the rhuMAb HER2 of 25mg/mL at the 5mM histidine, pH6.0,60mM trehalose, 0.01% polysorbas20 ^TMThe stability of reconstituted formulation after the middle lyophilizing

Time (natural law)	The percentage ratio of whole protein
	The percentage ratio of whole protein				22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃	22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃	0	100.0	100.0	100.0	100.0
14	ND	100.0	ND	100.0	0	100.0	100.0	100.0	100.0

31	99.3	99.7	100.0	100.0
31	99.3	99.7	100.0	100.0	61	100.0	ND	ND	ND

Sample is rebuild with 4.0mL or 20.0mL BWFI (1.1% or 0.9% benzyl alcohol), is deposited in 5 ℃ or 25 ℃ then.The percentage ratio of whole protein is defined as the component of non-degeneration peak area with non-degeneration size exclusion chromatographic determination.ND is not for measuring.

Table 6

Protein concentration be the rhuMAb HER2 of 25mg/mL at the 5mM histidine, pH6.0,38.4mM mannitol, 20.4mM sucrose, 0.01% polysorbas20 ^TMThe stability of reconstituted formulation after the middle lyophilizing

Time (natural law)	The percentage ratio of whole protein
	The percentage ratio of whole protein				22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃	22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃	0	99.7	99.7	99.8	99.8
14	ND	100.0	ND	99.8	0	99.7	99.7	99.8	99.8
14	ND	100.0	ND	99.8	31	100.0	100.0	100.0	100.0
92	100.0	ND	100	ND	31	100.0	100.0	100.0	100.0

Table 7

Protein concentration be the rhuMAb HER2 of 21mg/mL at the 10mM sodium succinate, pH5.0,250mM trehalose, 0.2% polysorbas20 ^TMThe stability of reconstituted formulation after the middle lyophilizing

Time (natural law)	The percentage ratio 21mg/mL protein of whole protein
	The percentage ratio 21mg/mL protein of whole protein		5℃	25℃
	0	99.8	5℃	25℃	99.8
14	0	99.8	ND	100.0	99.8
14	31	99.9	ND	100.0	99.4
92	31	99.9	99.8	ND	99.4

Sample is rebuild with 20.0mL BWFI (0.9% benzyl alcohol), is deposited in 5 ℃ or 25 ℃ then.The percentage ratio of whole protein is defined as the component of non-degeneration peak area with non-degeneration size exclusion chromatographic determination.ND is not for measuring.

As mentioned above, rhuMAb HER2 main degradation pathway desamidation or form butanimide in aqueous solution.Measure now in the rhuMAb HER2 preparation of four kinds of reconstructions because the non-denatured protein matter that desamidation or formation butanimide lose.

RhuMAb HER2 desamidation and formation butanimide can be analyzed with cation-exchange chromatography.(4.6 * 250mm) flow velocity is 1mL/min to Bakerbong Wide-Pore Carboxy Sulfon (CSX) post.The mobile phase buffer is (A) 0.02M sodium phosphate, pH6.9 and (B) 0.02M sodium phosphate, pH6.9,0.2M NaCl.Chromatograph is carried out as follows at 40 ℃:

Table 8

Time (minute)	The percent of buffer B
Time (minute)	The percent of buffer B	0	10
55	45	0	10
55	45	57	100
62	100	57	100
62	100	62.1	10
63	10	62.1	10

Eluting peak detects under 214nm, and each analysing protein applied sample amount is 75 μ g.

Equally, the stability of formulation that is reconstructed into 22mg/mL and 100mg/mL protein concentration does not have difference (Fig. 5 to 7) yet.Every kind of preparation is faster than 5 ℃ 25 ℃ protein degradation speed, and all degradation speeds that are deposited in 5 ℃ preparation are similar.Omit fast than succinate preparation at 25 ℃ of degradation speeds that contain the preparation of histidine.Trehalose concentration under two temperature in the preparation to degradation speed all less than the influence (Fig. 5 to 8).These results prove that the degradation speed of (5 ℃) is an acceptable to these four kinds of preparations under refrigerated condition in (in 30 days after rebuilding with BWFI) between demand usage time.

Muptiple-use use preparation will use in the U.S. must be by testing as the described anti-corrosive properties of American Pharmacopeia (USP).To contain 25mg/mL protein, 5mM histidine, pH6.0,60mM trehalose, 0.01% polysorbas20 ^TMRhuMAb HER2 lyophilized formulations be that 0.9 to 1.5% benzyl alcohol is rebuild with 20mL concentration w/w.When concentration was equal to or greater than 1.3%w/w, it is muddy that reconstituted formulation becomes after being incubated overnight under the room temperature (～25 ℃).BWFI preparation (0.9% benzyl alcohol) reconstruction with standard will make solution not test by anti-corrosive properties all the time.Yet, can test by anti-corrosive properties with the preparation that benzyl alcohol 1.0 or 1.1% and the compatible concentration of preparation is rebuild.The specification requirement solution concentration of product is in ± 10% scope, so lyophilized formulations is rebuild with 1.1% benzyl alcohol (1.1 ± 0.1%).

Studied a kind of rhuMAb HER2 preparation single step freezing dry process now.In the single step freezing dry process, the rhuMAb HER2 of 25mg/mL, 60mM trehalose, 5mM histidine (pH6) and 0.01% polysorbate20 are that 20 ℃, pressure are lyophilizing under the condition of 150mTorr in the shelf temperature.After 47 hours, the residual water content of lyophilized cake is less than 5%.This freeze-drying method is considered to favourable, because it has removed the secondary drying step, has simplified production process.

Embodiment 2

Anti-IgE preparation

IgE antibody can combine with the specificity high-affinity receptor on the mastocyte, cause the mastocyte threshing and discharge amboceptor as producing the histamine of allergy symptoms, therefore, hinder IgE and the bonded anti-IgE antibodies of high-affinity receptor and very high medical value is arranged for the treatment allergic disease.These antibody can not combine with IgE after IgE and receptors bind, because this will cause the release of histamine.This example has been described the exploitation to the lyophilized formulations that contains the total length humanization anti-IgE antibodies MaE11 (J.Immunology, 151:2623-2632 (1993)) that describes just like people such as Presta.

Material. highly purified rhuMAb E25 (the humanized anti-IgE antibodies MaE11 of reorganization), do not contain polysorbas20 in following formulations employed ^TM(Los Angeles CA) buys Spectra/Por 7 dialyzers from Spectrum.Other all reagent can and be AG from commercial acquisition in this research.Preparation buffer and chromatogram flow phase can be by in volumetric flask, mixing appropriate amount buffer and salt and Milli-Q level water make.

Preparation: E25 S agarose aggregation is dialysed in the specific formulation buffer.Dialysis realized 2-8 ℃ of exchange with the minimum exchange buffering liquid of 4 * 2L in 48 hours.After dialysis, freeze drying protectant added in some preparations as required form isotonic concentration.Protein concentration after the dialysis is measured with the ultraviolet spectrophotometer of 1.60 molar absorption coefficients.Protein after the dialysis is diluted to predetermined formulation concentrations with the appropriate formulation buffer, carries out aseptic filtration with 0.22 μ m Millex-GV filter (Millipore), branch installs in advance in the vial that cleaning and autoclaving cross then.The polytetrafluoroethylene bottle stopper of silanization, lyophilization under following condition then on the small bottle packing: the E25 preparation is refrigerated to-55 ℃ with 80 ℃/hour speed, and keeps the interior material of bottle freezing 4 hours.Then temperature is risen to 25 ℃ with 10 ℃/hour speed and carry out elementary drying.Elementary drying was carried out 39 hours under 25 ℃, 50 μ container constant pressures (chamer vacuumpressure), made that the residual water content in the lyophilized cake is 1-2%.After lyophilization, bottle of every kind of preparation is taken out, carry out t=0 and analyze, remaining bottle then different temperature as-70 ℃, 2-8 ℃, 25 ℃, 30 ℃ (control room temperature), 40 ℃ and 50 ℃ analyze.

Chromatograph: at Bio-Rad Bio-Select ^TMSEC 250-5 post (carries out non-degeneration size exclusion chromatograph on 300 * 7.8mm).Post is washed with the flow velocity equiulbrium flow of 0.5mL/min with PBS, and uses the Hewlett Packard 1090L type HPLC instrument that diode arrangement detector is housed.(Bio-Rad Inc.) proofreaies and correct post to use the molecular weight standard product of being made up of Elityran (670kd), gamma Globulin (158kd), ovalbumin (44kd) and vitamin B12 (1.35kd).The sample applied sample amount is 25 μ g, and protein is by (PE Nelson, Inc.) the ultraviolet light absorption value under the monitoring 214nm obtains with Turbochrom 3 softwares.

Hydrophobic interaction chromatography: with TosoHass Butyl-NPR post (3.5 * 4.6mm) and the F (ab ') that diode is arranged the Hewlett Packard 1090L type HPLC instrument chromatograph E25 antibody of detector is housed ₂Fragment.Elution buffer A is: 20mM Tris, 2M ammonium sulfate, 20% (v/v) glycerol, and pH8.0, and elution buffer B is 20mM Tris, 20% (v/v) glycerol, pH8.0.Post with 10% elution buffer B with the flow velocity balance of 1.0mL/min at least 20 minutes.The sample applied sample amount is 5 μ g, and protein is by (PE Nelson, Inc.) the ultraviolet light absorption value under the monitoring 214nm obtains with Turbochrom 3 softwares.After injecting sample, post was kept in 10% buffer B 1 minute, in 20 minutes, use the buffer B eluting of from 10% to 62% linear gradient then.Washed post 5 minutes with 100% buffer B, then between continuous sample injects with 10% buffer B balance again at least 20 minutes.

Antibody binding activity: sample is carried out IgE receptors bind inhibition mensuration (IE25:2) as the method that people such as top Presta describe, sample is with measuring diluent (phosphate buffer, 0.5%BSA, 0.05% polysorbate20,0.01% thiomersalate (Thimerosol)) is diluted to 20 μ g/mL and 30 μ g/mL.Every kind of diluent of replication is three times then, and the result is multiplied by the suitable dilution coefficient more just can generate active concentration.The result of average 6 mensuration.Thereby this algoscopy has been measured rhuMAb E25 competitiveness and has been stoped IgE and the bonded ability of high-affinity receptor that is fixed on the elisa plate in conjunction with IgE.The result can obtain than the value of living divided by the antibody concentration that ultraviolet light absorption value optical spectroscopy records.Previous test shows that this measurement result can represent stability.

Granulating test: merge the reconstituted formulation of lyophilizing rhuMAb E25, obtain volume and be about 7mL.Measure the granule number of particle diameter between 2 to 80 μ m in the 1mL sample with Hiac/Royo type 8000 enumerators.Enumerator is at first given a baby a bath on the third day after its birth inferior with the 1mL sample, replication 1mL sample is three times then.Be equal to or greater than the granule number of particle diameter 10 μ m and the granule number that particle diameter is equal to or greater than 25 μ m in every milliliter of the Instrument measuring.

The first step of preparation anti-IgE antibodies is buffer and a pH value suitable when determining product lyophilizing and preservation.With concentration is that the antibody of 5.0mg/mL is formulated in sodium phosphate, potassium phosphate and the histidine buffering liquid of the 10mM succinate buffer of pH5.0 to 6.5 and pH7.0.Fig. 9 be illustrated in before the lyophilizing and lyophilizing after in the preparation of higher pH antibody condensation product amount increase is arranged.The histidine preparation that exception is pH7 finds that its condensation product amount when 2-8 ℃ of preservation does not increase.Figure 10 represent rhuMAb E25 in the lyophilizing of the 5mM of pH6 and pH7 histidine buffering liquid then 2-8 ℃, 25 ℃ and the cohesion situation of 40 ℃ of preservations after 1 year.The condensation product amount of the preparation of pH6 is that 7 antibody preparation lacks than pH all under each minute point and preservation temperature.This result shows that the histidine of pH6 is particularly suitable for as the Laemmli buffer system Laemmli that prevents the antibody cohesion.

Be to realize the screening of freeze drying protectant, can be when being with or without freeze drying protectant anti-IgE antibodies be formulated into pH and be in 5 the sodium succinate.The effective freeze drying protectant that adds to isotonic concentration can be divided into 3 classes:

(a) irreducibility monosaccharide (being mannitol);

(b) reducing disaccharides (being lactose and maltose); With

(c) non-reducing disaccharide (being trehalose and sucrose).

Preparation is presented in Figure 11 and 12 2-8 ℃ and the cohesion situation of 40 ℃ of preservations after 1 year.When 2-8 ℃ of preservation, identical in the contrast of the cohesion speed of monosaccharide preparation (mannitol) and buffer, and the preparation that contains disaccharide aspect the control cohesion all very effectively (Figure 11).Result 40 ℃ of preservations is identical, just the cohesion of sucrose preparation rapid (this be associated with lyophilized cake generation browning reaction (Figure 12)).The back will show that this is that sucrose degraded takes place during preservation and causes under acid pH and high temperature.

The hydrophobic interaction chromatography that is formulated into the histidine buffering liquid of pH6 and the antibody in the lactose shows that antibody is at 40 ℃ of preservations will change after 6 months (Figure 13).Chromatographic peak broadens and the retention time minimizing.And shown in Figure 14 and 15, these variations are not then found in buffer contrast and sucrose preparation preservation under condition of similarity.And the antibody pI that is formulated into lactose that isoelectrofocusing is presented at 25 ℃ and 40 ℃ preservations has acid moving.This shows the uncomfortable freeze drying protectant as antibody of reducing sugar.

Figure 16 has represented that concentration is the cohesion situation of lyophilized formulations after 50 ℃ of 12 weeks of preservation of the anti-IgE of 20mg/mL in the sucrose of the 5mM of pH6 histidine buffering liquid and various concentration and trehalose.When the sugared concentration of every mole of antibody during greater than 500 moles, the effect of condensing that prevents of two kinds of sugar is similar.According to these results, to sucrose and trehalose etc. blend hypertonic preparation and further study.Preparation is added into the antibody of low concentration before lyophilizing, freeze dried product is used than the consumption amount still less of the injection antibacterial (BWFI) that contains 0.9% benzyl alcohol and rebuild.This makes the effect between protein and antiseptic when antibody can concentrate and comprise that antiseptic has been avoided long-time preservation with the preparation that is used for muptiple-use use before subcutaneous defeated passing.

Deng oozing preparation: the concentration that is mixed with anti-IgE in the 5mM of pH6 histidine buffering liquid is 25mg/mL, and every mole of antibody sugar molal quantity is 500 moles, and promptly sugared concentration equals 85mM.This preparation BWFI littler 4 times than original dosage volume (0.9% benzyl alcohol) rebuilds.Antibody concentration is 100mg/mL in this 20mM histidine that makes at pH6, waits that to ooze sugared concentration be 340mM.

Hypertonic preparation: the concentration that is mixed with anti-IgE in the 5mM of pH6 histidine buffering liquid is 25mg/mL, and every mole of antibody sugar molal quantity is 1000 moles, and promptly sugared concentration equals 161mM.This preparation BWFI littler 4 times than original dosage volume (0.9% benzyl alcohol) rebuilds.Antibody concentration is 100mg/mL in this 20mM histidine that makes at pH6, and hyperosmotic glucose concentration is 644mM.

Deng blending relatively being presented among Figure 17 to 19 of the antibody cohesion of hypertonic preparation after preservation is up to 36 weeks.The height that is deposited in 2-8 ℃ ooze or etc. ooze in the preparation and to find that all cohesion changes (Figure 17).And be deposited in control room temperature (30 ℃) following time, do not find to have the cohesion increment in the hyperosmotic solution, and ooze 1 to 2% the condensation product increment (Figure 18) of having an appointment in the preparation waiting.At last, when 50 ℃ of preservations, finding in hypertonic preparation has less condensation product increment, 4% condensation product increment is arranged in the isotonicity trehalose preparation, and 12% condensation product increment (Figure 19) is arranged in the isotonicity sucrose preparation.These results show that the isotonicity preparation should contain the sugar that makes keep stablizing necessary minimum up to the antibody capable of 30 ℃ of preservations.

With the IgE receptor suppress that algoscopy can be measured and wait to blend anti-IgE in the hypertonic preparation in conjunction with activity.Find to wait and to blend height and ooze sucrose and trehalose preparation and after-70 ℃, 2-8 ℃, 30 ℃ and 50 ℃ of 36 weeks of preservation, do not change basically in conjunction with activity.

Contain insoluble condensation product or granule (people such as Cleland, CriticalReviews in Therapeutic Drug Carrier Systems, 10 (4): 307-377 (1993)) in the known lyophilized formulations.Therefore, to adding the pH6 of 85mM and 161mM sucrose and trehalose, the concentration in the histidine of 5mM is carried out granulating for the 25mg/mL lyophilized antibodies and is measured.Adding polysorbate20 to concentration is 0.005%, 0.01% and 0.02%.The lyophilizing sample also is being reconstructed into the 20mM histidine, and antibody concentration is 100mg/mL among the pH6, and sugared concentration is 340mM and 644mM.The concentration of rebuilding the back polysorbate20 is respectively 0.02%, 0.04% and 0.08%.

Following table 9 expression waits and blends that particle diameter is equal to or greater than the granule number of 10 μ m and the granule number that particle diameter is equal to or greater than 25 μ m in sucrose that height oozes and the trehalose preparation.Before lyophilizing, polysorbate20 added in the preparation to concentration be 0.005%, 0.01% and 0.02%.The result shows and adds tween (Tween) in the preparation ^TMCan significantly reduce the granule number in each test particle size range.American Pharmacopeia (USP) for the standard of little injected dose is: particle diameter is no more than 6000 more than or equal to the granule number of 10 μ m in each container, and particle diameter is no more than 600 (people such as Cleland, the same) more than or equal to the granule number of 25 μ m.After adding polysorbate20, height blends etc. and to ooze preparation and all passed through test.

Table 9

Preparation	Polysorbate20	Every milliliter granule number
		Every milliliter granule number		≥10μm	≥25μm
		Isotonicity sucrose	No	≥10μm	≥25μm	16122	28
	0.005％	Isotonicity sucrose	No	173	2	16122	28
	0.005％		0.01％	173	2	224	5
	0.02％		0.01％	303	6	224	5
	0.02％	Hypertonicity sucrose	No	303	6	14220	84
	0.005％	Hypertonicity sucrose	No	73	6	14220	84

	0.01％	51	0
	0.01％	51	0	0.02％		6
The isotonicity trehalose	No	33407	24	0.02％		6
The isotonicity trehalose	No	33407	24	0.005％	569	4
	0.01％	991	16	0.005％	569	4
	0.01％	991	16	0.02％	605	9
The hypertonicity trehalose	No	24967	28	0.02％	605	9
The hypertonicity trehalose	No	24967	28	0.005％	310	11
	0.01％	209	6	0.005％	310	11
	0.01％	209	6	0.02％	344	6

Following table 10 has shown a kind of preparation (that is, the bottled rhuMAb E25 of 143mg isotonicity preparation) of anti-IgE antibodies, and it is considered to be applicable to subcutaneous defeated the passing of this antibody.Adding 5.7mL concentration in the 10cc bottle is the rhuMAb E25 of 25mg/mL, and rhuMAb E25 is formulated in the 5mM histidine and 0.01% polysorbate20 of pH6.It is 85mM that sucrose is added to concentration as freeze drying protectant, and corresponding sugar is 500 to 1 with the mol ratio of antibody.Bottle is carried out lyophilization, be reconstructed into 1/4 or 1.2mL of original dosage volume with 0.9% benzyl alcohol.The final concentration of formulation components increases by 4 times, and rhuMAb E25 concentration is 100mg/mL in the 20mM histidine of formation pH6, and polysorbate20 is 0.04%, and sucrose is the benzyl alcohol of 340mM (isotonicity) and 0.9%.Preparation contains the histidine buffering liquid of pH6, can prevent the antibody cohesion because proved it.Adding sucrose is because the use in the medicine industry in the past as freeze drying protectant.The selection of sugar concentration should make that when rebuilding be the isotonicity preparation.At last, can add the formation that polysorbate20 prevents insoluble condensation product.

Table 10

Preparation (dosage is 5.7mL in the 10cc bottle) before the lyophilizing	Reconstituted formulation (1.2mL 0.9% benzyl alcohol)
	Reconstituted formulation (1.2mL 0.9% benzyl alcohol)	25mg/mL rhuMAb E25	100mg/mL rhuMAb E25
The 5mM histidine, pH6.0	The 20mM histidine, pH6.0	25mg/mL rhuMAb E25	100mg/mL rhuMAb E25
The 5mM histidine, pH6.0	The 20mM histidine, pH6.0	85mM sucrose	340mM sucrose
0.01% polysorbate20	0.04% polysorbate20	85mM sucrose	340mM sucrose
0.01% polysorbate20	0.04% polysorbate20	-	0.9% benzyl alcohol

Claims

1. a preparation comprises the anti-IgE antibodies that content is 5-40mg/mL, and content is sucrose or trehalose, buffer and the surfactant of 80-300mM.

2. preparation according to claim 1, it is freeze dried and stablized at least 1 year under 30 ℃ of conditions.

3. preparation comprises the anti-IgE antibodies of 2-50mg/mL, and content is the sugar of 10-400mM and the freeze-dried mixture of buffer.

4. each preparation of claim 1-3, wherein said buffer is histidine, phosphate, Tris, citrate, succinate or other organic acid buffer.

5. the preparation of claim 4, wherein said buffer is a histidine buffering liquid.

6. each preparation of claim 1-5 also comprises surfactant.

7. the preparation of claim 6, wherein said surfactant is a nonionic surfactant.

8. the preparation of claim 7, wherein said surfactant is a polysorbate.

9. each preparation of claim 1-8, the concentration of wherein said anti-IgE antibodies is about 20-30mg/mL.

10. each preparation of claim 1-9, the content of wherein said sucrose or trehalose is about 80-170mM.

11. each preparation of claim 1-10, it is a lyophilized form.

12. a bottle, it comprises the preparation of claim 11.

13. a reconstituted formulation, it is by with each formulation preparation of diluent dilution claim 1-11.

14.。The reconstituted formulation of claim 13, the concentration of anti-IgE antibodies is equal to or greater than 50mg/mL in the wherein said reconstituted formulation.

15. the reconstituted formulation of claim 14, the concentration of anti-IgE antibodies is about 15-45mg/mL in the wherein said reconstituted formulation.

16. the reconstituted formulation of claim 15, the concentration of anti-IgE antibodies is about 15-25mg/mL in the wherein said reconstituted formulation.

17. each reconstituted formulation of claim 13-16, wherein said diluent is selected from: sterilized water, injection antibacterial (BWFI), pH buffer, aseptic salt solution, Ringer's solution or Glucose Liquid.

18. each preparation of claim 1-11,13-17 has purposes in this patient's who needs the medicine in preparation treatment.

19. the purposes of claim 18, the concentration of anti-IgE antibodies is equal to or greater than 50mg/mL in the wherein said reconstituted formulation.

20. the purposes of claim 19, the concentration of anti-IgE antibodies is about 15-45mg/mL in the wherein said reconstituted formulation.

21. the purposes of claim 20, the concentration of anti-IgE antibodies is about 15-25mg/mL in the wherein said reconstituted formulation.